Article Negligible interchromosomal effect in embryos of Robertsonian translocation carriers

RBMOnline - Vol 10. No 3. 2005 363-369 Reproductive BioMedicine Online; www.rbmonline.com/article/1630 on web 17 January 2005 Article Negligible interchromosomal effect in embryos of Robertsonian translocation carriers Dr Santiago Munné Santiago Munné has been director of PGD at Saint Barnabas Medical Center since 1995. His group there focuses on identifying genetically normal embryos. Originally from Barcelona, Spain, Dr Munné gained his PhD in genetics from the University of Pittsburgh and joined Dr Jacques Cohen at Cornell University Medical College, New York in 1991. There he developed the first PGD test to detect embryonic numerical chromosome abnormalities. His work has been recognized by several prizes: in 1994, 1995 and 1998 from the Society for Assisted Reproductive Technology, and in 1996 from the American Society for Reproductive Medicine. Recently the PGD team has shown higher pregnancy rates in women of advanced age undergoing PGD. This team has performed more than 250 PGD cycles for translocations and over 2100 PGD cycles for chromosome abnormalities related to advanced maternal age. Dr Munné has more than 100 publications to his name, and is a frequent lecturer, both nationally and internationally, on his team s work and the field of preimplantation genetics. They recently were blessed with their first daughter, Mar. Santiago Munné 1,5, Tomas Escudero 1, Jill Fischer 1,2, Serena Chen 2, Joseph Hill 3, James R Stelling 4, Anna Estop 1 Reprogenetics, LLC, West Orange, NJ, USA; 2 The Institute of Reproductive Medicine and Science, Livingston, NJ, USA; 3 Fertility Centres of New England, 20 Pond Meadow Drive, Suite 101, Reading, MA, USA; 4 Reproductive Specialists of NY, Mineola, NY, USA 5 Correspondence: e-mail: munne@embryos.net Abstract It has been suggested that translocations, and perhaps other chromosome rearrangements, disturb meiotic disjunction of chromosome pairs not involved in the translocation, resulting in non-disjunction in those chromosomes (interchromosomal effect) and predisposition to trisomy offspring. Other reports have suggested an increased risk of mosaicism and chaotic embryos in translocation carriers. This study was designed to determine if such interchromosomal effects are producing significantly more chromosome abnormalities than those expected from unbalanced gametes. For that purpose, two groups of PGD patients were compared, Robertsonian translocation carriers (RBT) and carriers of X-linked diseases (XLI), of similar age. Both groups were analysed by FISH with similar DNA probes. The results indicate that overall, the higher rate of chromosome abnormalities in the RBT group was solely due to unbalanced gametes and not to an interchromosomal effect or higher incidence of mosaicism. If unbalanced and normal were combined, this proportion was 53% in XLI and 59% in RBT. However, when specific RBT translocations were studied, only a slight increase in embryos with aneuploidy for chromosome 22 was found for the t(13;14) translocation carriers, while a higher rate of post-zygotic abnormalities was observed in the more rare RBT. In conclusion, the overall rate of non-translocation related abnormalities was not increased in the RBT group compared with the control group, but a slight interchromosomal effect may exist, as some Robertsonian translocations may be more prone to produce mosaic and chaotic embryos. Keywords: mosaicism, PGD, t(13;14), t(13;22), t(14;15), t(14;21) Introduction Preimplantation genetic diagnosis (PGD) is offered to carriers of translocations as an early form of prenatal diagnosis and an alternative to pregnancy termination of unbalanced fetuses. PGD for translocations has been attempted using a variety of fluorescence in-situ hybridization (FISH) methods, using chromosome painting probes in polar bodies, breakpointspanning probes, combination of telomeric and centromeric probes, or different methods to convert or detect metaphase stage chromosomes (Munné et al., 1998a,b,c,d, 2000a,b, Conn et al., 1998; Durban et al., 1998; Verlinsky et al., 1999; Willadsen 1999; Evsikov et al., 2000; Tanaka et al., 2004). By selecting only normal and balanced embryos after PGD for replacement, these patients have a higher chance of conceiving and a reduced probability of spontaneous abortions and chromosomally unbalanced offspring (Munné et al., 1998b, 2000a; Cieslak et al., 2003). However, there is a very strong inverse correlation between chromosomal abnormality and pregnancy rates (Munné et al., 2000a; Verlinsky et al., 2002), and several reports on PGD for translocations have showed high rates of abnormal embryos, with few resulting in pregnancy after PGD (Conn et al., 1998; Van Asche et al., 1999; Verlinsky et al., 2002; Gianaroli et al., 2003). Cases involving Robertsonian translocations achieve 363

364 significantly higher pregnancy rates than cases involving reciprocal translocations, presumably because they produce lower numbers of unbalanced embryos (Munné et al., 2000b; Gianaroli et al., 2003). In male carriers, this is due to a lower number of unbalanced spermatozoa (Egozcue et al., 2003). However, in addition to unbalanced embryos for the chromosomes involved in the translocation, there is also the possibility that other chromosomes may be also abnormal in higher proportions than would be expected for these patients. It has been suggested that translocations, and perhaps other chromosome rearrangements, disturb meiotic disjunction of chromosome pairs not involved in the translocation, resulting in non-disjunction for these chromosomes (an interchromosomal effect) and predisposition to trisomy offspring (Lindenbaum et al., 1985). Epidemiological studies (Aurias et al., 1978; Lindenbaum et al., 1985) have suggested the possible relationship between the presence of a balanced reorganization and trisomy 21, although other studies have not confirmed this (Warburton, 1985; Schinzel, 1992). The ideal method to detect the existence of any interchromosomal effect is by analysing sperm cells, where negative selection due to lethal segregation or other abnormalities has not yet affected the survival of the embryo. Sperm analyses of male translocation carriers have shown contradictory results. Some studies have found no effect (Martin, 1988; Pellestor, 1990; Martin et al., 1992; Syme and Martin, 1992; Estop et al., 1996, 2000; Templado et al., 1996; Van Hummelen et al., 1997; Martini et al., 1997; Blanco et al., 1998, 2000; Cifuentes et al., 1999; Honda et al., 1999) while other studies have detected an interchromosomal effect (Rousseaux et al., 1995; Mercier et al., 1998; Blanco et al., 2000; Morell et al., 2001; Oliver- Bonet et al., 2001; Pellestor et al., 2001; Shi and Martin, 2001). For patients undergoing PGD of translocations, it is important to know if their risk of abnormalities for chromosomes not related to the translocation is high enough to require the simultaneous analysis of other chromosomes. For that purpose, two groups of PGD patients were compared, Robertsonian translocation carriers (RBT) and carriers of X-linked diseases (XLI), in order to determine if an excess of mosaic and/or aneuploid embryos other than those derived from the meiotic segregation of the Robertsonian translocation was detected in the RBT group. Materials and methods Patients Two groups of patients were included in this study, those undergoing PGD for Robertsonian translocations (RBT group) and those undergoing PGD for X-linked monogenic diseases but with normal karyotypes (XLI group). Only patients 35 and younger were included in the study, to minimize the effect of maternal age. Patients included in this retrospective study underwent IVF, embryo biopsy and cell fixation in 17 different centres all referring the fixed cells for PGD analysis to Reprogenetics, LLC. Of all RBT cycles, 31 were performed at the Institute for Reproductive Medicine and Science (Livingston, NJ, USA), seven at the Fertility Centre of New England (Reading, MA, USA), six at Reproductive Specialists of NY (Mineola, NY, USA) and the remaining among the other centres. Institutional review board consents for PGD were signed for each patient at each centre. Embryo biopsy, fixation, and FISH During day 3 of development, one cell per embryo was biopsied by zona drilling using acidified Tyrode s solution or laser, and the embryos returned to culture as described elsewhere (Munné et al., 2003a). All of the embryos were at the 4- to 12-cell stage at the time of biopsy. All blastomeres were fixed individually using Carnoy s solution (Velilla et al., 2002). FISH was performed using two successive hybridizations, the first one with probes for 13, 16, 18, 21 and 22 chromosomes, and the second with X, Y, 15 plus either 1, 14 or 17, after previously published methods (Munné et al., 2003b). In some patients with RBT involving chromosome 14, the second panel did not include chromosomes X and Y. All the chromosomes involved in Robertsonian translocations were included in these sets of FISH probes; therefore, all patients in both groups had the same chromosomes analysed in the first panel, and similar ones in the second. Because only a small fraction of embryos classified by PGD as chromosomally abnormal could be fully reanalysed due to time constraints, criteria previously described were followed (Munné et al., 2004) to classify embryos as aneuploid, polyploid, haploid or mosaic based on single cell analysis. Because all embryos included in the study were obtained from M-II oocytes showing a clear polar body, and after syngamy, all zygotes included in the study showed two pronuclei and two polar bodies, abnormalities such as polyploidy and haploidy were considered to be mostly post-zygotic (Munné et al., 1994) although a tiny fraction could still be produced by diploid spermatozoa (0.13%; Wyrobek et al., 1994). Statistical analysis Since the patients contributed a variable number of cycles to the data, it was first necessary to examine the error structure, to test whether the between patient error was significantly greater than that within patient. If not the case, the investigation could be carried through using one of the many procedures available for comparing proportions; for example, the chi-squared test, or Fisher s exact test. If, however, there were evidence of heterogeneity, a more complex generalized linear model (GLM) analysis would be necessary. Failure to observe this procedure usually results in an exaggeration of the importance of any effect being investigated. Although the GLM analysis operates on the logistic scale, the results are presented on the original scale of percentages. Results The RBT group consisted of 55 patients who underwent 76 cycles of PGD, of which 54 cycles were carriers of

45,der(13;14)(q10;q10), eight of 45,der(14;21)(q10;q10), four of 45,der(14;15)(q10;q10), three of 45,der(13;22)(q10;q10), four of 45,der(13;15)(q10;q10), two of 45,der(14;22) (q10;q10), and one of 45,der(13;21)(q10;q10). Of these cycles, 35 were female and 41 male carriers. The XLI group consisted of 47 patients who had 52 cycles of PGD for X-linked monogenic diseases, and all of them had normal karyotypes. The average maternal age was 31.1 for the RBT group and 30.6 for the XLI group. The RBT group had 627 embryos biopsied of which 578 produced FISH results. The XLI group had 430 embryos biopsied, of which 412 could be analysed. In the remaining embryos, either the biopsied cells did not have a nucleus or they were not properly fixed and did not yield results. The XLI group had significantly (P < 0.001) more normal embryos (53.2%) than the RBT group (28.9%) and the difference was due entirely to an increase in aneuploidy produced by the Robertsonian translocation, and here referred as unbalanced embryos (Table 1). Indeed, if the data of the unbalanced embryos was added to the normal or balanced embryos in the RBT group, no statistical difference appeared for this subgroup (normal + balanced + unbalanced) (53.2% in XLI compared with 62.1% in RBT), and both groups had the same profile of diagnosis. The percentage of aneuploidy in XLI-derived embryos (18.2%) was similar to the percentage of aneuploidy for chromosomes unrelated to the translocations in the RBT group (10.6%) (Table 1). However, the 18.2% aneuploidy rate in the XLI group did contain those embryos with aneuploidies for chromosomes involved in the translocation, thus, a better method to compare aneuploidy rates is analysing one translocation type at a time, and excluding the chromosomes involved in the translocation in both XLI and RBT groups. This could be done only for the most common RBT, t(13;14). For t(13;14), individual aneuploidy rates for chromosomes XY, 16, 18, 21 and 22 were compared with those found in the XLI group (Table 2). Only a slight increase (P < 0.05) of aneuploidy for chromosome 22 was detected for the RBT group with t(13;14) when compared with the XLI group. For the other RBT types, the numbers were too small to be meaningful. Table 1. Numbers of chromosome abnormalities in Robertsonian translocation (RBT) and X-linked disease (XLI) embryos. Values in parentheses are percentages. FISH results XLI RBT P-value Patients (n) 47 55 Cycles (n) 52 76 Normal or balanced 219 (53.2) 167 (28.9) <0.001 Unbalanced a 0 192 (33.2) Unbalanced a and aneuploid 0 10 (1.7) Aneuploid 75 (18.2) 61 (10.6) Post-meiotic abnormalities b 118 (28.6) 148 (25.6) Total (n) 412 578 Normal + unbalanced 219 (53.2) 359 (62.1) NS Combined aneuploid 75 (18.2) 71 (12.3) NS NS = not significant. a Unbalanced are those embryos that are aneuploid for the chromosomes involved in the specific Robertsonian translocation carried by the couple. b Including haploid, polyploid, chaotic and complex abnormal embryos. If the embryos were reanalysed and more than one cell was available, it also included mosaic embryos. Table 2. Number of aneuploidy events a for chromosomes 16, 18, 21 and 22 in t(13;14) and X-linked diseases (XLI). Values in parentheses are percentages. Indication No. Chromosome embryos 16 18 21 22 XY t(13;14) 411 23 (5.6) 12 (2.9) 13 (3.2) 17 (4.1) 2 b (1.1) XLI 412 15 (3.6) 12 (2.9) 18 (4.3) 6 (1.5) 10 (2.4) P-value NSNS NS 0.05 NS XLI = X-linked diseases; NS = not significant. a Embryos with two aneuploidies were counted as two different events. b Only 186 out of 411 embryos were analysed with XY probes. 365

Table 3. Number of chromosome abnormalities in Robertsonian translocations according to translocation types. Values in parentheses are percentages. FISH results t(13;14) t(14;21) Other P-value c Patients (n) 41 5 9 Cycles (n) 54 8 14 Normal or balanced 129 (31.3) 13 (27.1) 25 (21.2) <0.05 Unbalanced a 140 18 34 Unbalanced a and aneuploid 8 2 0 Aneuploid only 48 1 12 Post-meiotic abnormalities b 87 (21.1) 14 (29.2) 47 (39.8) <0.001 Total (n) 412 48 118 Normal or balanced + unbalanced 269 (65.3) 31 (64.6) 59 (50.0) NS Total aneuploid 56 (13.6) 3 (6.3) 12 (10.2) NS Total unbalanced a 148 (35.9) 20 (41.7) 34 (28.8) NS NS = not significant. a Unbalanced are those embryos that are aneuploid for the chromosomes involved in the specific Robertsonian translocation carried by the couple. b Including haploid, polyploid, chaotic and complex abnormal embryos. If the embryos were reanalysed and more than one cell was available, it also included mosaic embryos. c The P-value relates to a comparison of t(13;14) and t(14;21) combined, versus other. Table 4. Number of chromosome abnormalities in Robertsonian translocations according to sex of the carrier. Values in parentheses are percentages. FISH results Female Male P-value Patients (n) 25 30 Cycles (n) 35 41 Normal or balanced 79 (28.4) 88 (29.3) NS Unbalanced a 94 98 Unbalanced a and aneuploid 2 8 Aneuploid only 27 34 Post-meiotic abnormalities b 76 (27.3) 72 (24.0) NS Total (n) 278 300 Normal or balanced + unbalanced 173 (62.2) 186 (62.0) NS Total aneuploid 29 (10.4) 42 (14.0) NS Total unbalanced a 96 (34.5) 106 (35.3) NS NS = not significant. a Unbalanced are those embryos that are aneuploid for the specific Robertsonian translocation carried by the couple. b Including haploid, polyploid, chaotic and complex abnormal embryos. If the embryos were reanalysed and more than one cell was available, it also included mosaic embryos. 366 There was a similar rate of post-meiotic abnormalities for both groups when all translocations were pooled together (Table 1). As described in Table 3, if the RBT cycles were compared according to their translocation, when the analysis was performed on a per embryo basis, those cycles with Robertsonian translocation that were not t(13;14) nor t(14;21) had highly significant more post-meiotic abnormalities (39.8%) than those with t(13;14) (21.1%) (P < 0.001). Because the majority of cycles were t(13;14), the overall results for the RBT group are similar than those for the t(13;14) sub-group. Another difference between translocations is the range of chromosome abnormalities between patients. The t(13;14) had an average fraction of normal embryos of 0.30 ± 0.19 (range 0 0.83), and of aneuploidy embryos of 0.14 ± 0.14 (range 0 0.5) while the t(14;21) was more homogeneous with an average fraction of normal embryos of 0.2 ± 0.14 (range 0 0.33) and of aneuploidy embryos of 0.03 ± 0.09 (range 0 0.25). In contrast, when the RBT cycles were sorted according to gender of the carrier (Table 4), no significant differences were observed between male and female carriers regarding types of chromosome abnormalities and overall rate of chromosome abnormalities.

Discussion Some sperm FISH studies from translocation carriers have suggested the presence of interchromosomal effects for some translocations (Rousseaux et al., 1995; Mercier et al., 1998; Blanco et al., 2000; Morel et al., 2001; Oliver-Bonet et al., 2001; Shi and Martin, 2001), whereas others have not detected any such effects in other carrier patients (Van Hummelen et al., 1997; Martini et al., 1997; Blanco et al., 1998, 2000; Cifuentes et al., 1999; Honda et al., 1999; Estop et al., 2000). The interchromosomal effects detected in spermatozoa are small compared with the high rate of chromosome abnormalities detected in cleavage-stage human embryos (Munné et al., 1995; Marquez et al., 2000; Magli et al., 2001; Bielanska et al., 2002). To evaluate if interchromosomal effects significantly increase the rate of chromosome abnormalities in human embryos, PGD analysis on embryos of translocation carriers has been performed in a handful of studies, including the present one. The current work did not detect an overall interchromosomal effect on aneuploidy for chromosomes unrelated to the Robertsonian translocations studied. The rate of aneuploidy in the control group was similar to that seen in the RBT group. To rule out whether one chromosome had an excess of aneuploidy in the RBT group that could have been evened out inadvertently when pooling all chromosome aneuploidy together, individual rates of aneuploidy for chromosomes 16, 18, 21 and 22 were assessed in the most common RT, t(13;14). The findings showed find a slight significant increase (P < 0.05) of aneuploidy for chromosome 22 in the RBT group. A study by Conn et al. (1998) reported that over 50% of the embryos of two Robertsonian translocation carriers were chaotic mosaics. Unfortunately, the study did not provide a control group. Mosaics, and specifically chaotic mosaics have been linked to male factor infertility in karyotypically normal patients (Obasaju et al., 1999; Gianaroli et al., 2000, 2003; Silber et al., 2003), culture conditions (Munné et al., 1997), and in general to be patient-related (Delhanty et al., 1997). The results of the study indicate that although overall Robertsonian translocation carriers do not produce more post-zygotic abnormalities than controls, the more uncommon Robertsonian translocations may be linked to higher rates of post-zygotic abnormalities. The mechanism for such a phenomenon is not evident, and thus a larger sample of cases should be investigated, controlling for other factors, such male factor infertility and culture conditions. In a large sample of cases, these factors even out, as is the case of comparing all Robertsonian translocations with controls, but in smaller samples, other factors may play a big role biasing results. Another report describing a potential interchromosomal effect in cleavage stage embryos is by Gianaroli et al. (2002). They compared cycles of Robertsonian translocation cases with those of reciprocal translocations. Both groups were analysed for those chromosomes involved in the translocation in addition to at least chromosomes 13, 16, 18, 21 and 22. The maternal age of both groups was similar, 35 years, but higher than the present study (31 years for carriers and 30.6 for controls). Of the embryos of Robertsonian translocation cases 23% were normal, 44% were abnormal for the translocation with or without other abnormalities, and 33% had other abnormalities not related to the translocation. The Reciprocal translocation cases had much higher rates of abnormal embryos due to the translocation but lower rates of abnormalities involving non-translocation chromosomes. If the results obtained by Gianaroli et al. (2002) are compared with those in the present study, the proportion of normal embryos (23% in Gianaroli s and 29% in the present study), abnormal for the translocation with or without involving other chromosomes (44 and 35%) and abnormal for nontranslocation related chromosomes (33 and 36%), are similar, and where the slightly higher number of abnormal embryos in Gianaroli s study could be attributed to a higher maternal age. If the present control group is used to compare the results of both studies, it can be concluded that overall, there is no apparent interchromosomal effect that generates further abnormal embryos, with 60% (67/111) embryos abnormal for other chromosomes than the translocation in the Gianaroli et al. (2002) study, and 47% in the control group. The findings of Gianaroli et al. (2002) that reciprocal translocation cases have less chromosome abnormalities for those chromosomes not related to the translocation than Robertsonian translocations are worthy of attention. Cases involving Robertsonian translocations achieve significantly higher pregnancy rates than cases involving reciprocal translocations presumably because they produce lower numbers of unbalanced embryos (30 and 70% respectively; Munné, 2000b, this report, and unpublished data). Male reciprocal translocation carriers have more translocationrelated abnormal spermatozoa (47.5 81.0%) than Robertsonian carriers (3.4 36%) (Egozcue et al., 2003). Both Robertsonian and reciprocal translocations are found at a high incidence among infertile men presenting with oligozoospermia. This association is most marked in severe oligozoospermia. Meiotic pachytene configurations of male Robertsonian or reciprocal translocations have been seen to associate with the paired sex chromosomes forming the sex vesicle (Luciani et al., 1984) or other partially unpaired autosomal bivalents (Vidal et al., 1987), leading to spermatogenic arrest, and thus infertility or sterility. The sterilizing effect of translocations is limited to male meiosis. Moreover, it has been reported that synaptic anomalies of the reorganized chromosomes of carriers of structural rearrangements may lead to synaptic anomalies of other bivalents facilitating the non-disjunction of these chromosome pairs and possible interchromosomal effects (Egozcue et al., 1983; Goldman and Hulten, 1993). Neither meiotic studies nor sperm studies have provided conclusive evidence on whether one type of translocation (Robertsonian versus reciprocal) is more prone to meiotic interchromosomal effects. Recent reports suggest that the quality of the spermatozoa is a more significant factor in sperm aneuploidy, affecting both patients with normal karyotypes and those with translocations (Vegetti et al., 2000; Pellestor et al., 2001). Their data suggest that interchromosomal effects may be restricted only to carriers of structural rearrangements that are infertile and this association is strongest in men with oligoasthenozoospermia. Poor sperm quality may be the result of meiotic disturbances due to structural rearrangements or to a hormonally deficient testicular environment. There is a need for further studies in either sperm or embryos controlling for the type of 367

368 translocation, sperm quality parameters and FSH concentrations in order to shed further light on the relationship between translocations in males and interchromosomal meiotic effects. Indeed, because there are a limited number of Robertsonian translocations combinations, as opposed to reciprocal translocations, these intriguing questions should be addressed in the future, at least in Robertsonian carriers. Although the incidence of unbalanced offspring is more frequent for females than males (Boue and Galiano, 1984), in this study, similar rates of chromosomally abnormal embryos were found in males and females. According to Faraut et al. (2000), the observed sex ratio deviation may be related to male infertility caused by the translocation, reflecting an excess of maternal carriers reproducing compared with male carriers. In contrast, the preponderance of maternal transmission of a 3:1 imbalance was found to be associated with maternal age effect (Faraut et al., 2000). 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