Human Reproduction, Vol. 17, No. 11, 2957-2962,
November 2002
© 2002 European Society of Human Reproduction and Embryology
Correlation between fluorescence in-situ hybridization analyses and in-vitro development to blastocyst stage of embryos from Robertsonian translocation (13;14) carriers
1 Fertility Clinic Erasmus Hospital, French Speaking Free University of Brussels, Route de Lennik, 808, 1070 Brussels, 2 Medical Genetics and 3 Fertility Clinic and Laboratory of Biology and Psychology of Human Fertility, Free University of Brussels, French Speaking, Route de Lennik, 808, 1070 Brussels, Belgium
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BACKGROUND: Little is known about the extent and timing of selection against the embryos that are carriers of unbalanced translocations. METHODS: Fluorescence in-situ hybridization (FISH) with probes for chromosomes 13, 14 and 18 was performed, mostly on day 3, on 69 human embryos which were then allowed to develop further in culture to day 5, from five carriers of Robertsonian translocation (RT) t(13;14). RESULTS: Twelve normal/balanced blastocysts were replaced in seven consecutive cycles (day 5). Three cycles resulted in clinical pregnancies. The proportion of blastocysts displaying a normal/balanced karyotype was 56%, while only the 20% of blocked embryos were normal/balanced (
2: P < 0.05). All the embryos analysed on day 5, except one, displayed mosaicism. The percentages of diploid cells for chromosomes 13 and 14 were significantly lower than for chromosome 18 (chromosome 13: 49.0 ± 28.0; chromosome 14: 53.0 ± 31.8; chromosome 18: 75.7 ± 20.4; Mann–Whitney test: P < 0.01). The embryos displaying 
62% of diploid cells for at least two of the three chromosomes analysed, more frequently reached the blastocyst stage (blocked embryos: blastocysts chromosome 13: 43.1 ± 30.3, 64.9 ± 29.0; chromosome 18: 64.9 ± 29.0, 83.0 ± 12.9; Mann–Whitney test: P < 0.01). CONCLUSIONS: Normal/balanced embryos developed better but the proportion of abnormal blastocysts was still high. Preimplantation genetic diagnosis is recommended to select normal/balanced embryos from RT t(13;14) carriers.
Key words: blastocyst/FISH/human embryo/preimplantation genetic diagnosis/Robertsonian translocation
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Introduction |
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Robertsonian translocations (RT) are exchanges occurring close to the centromere of two acrocentric chromosomes (chromosomes 13, 14, 15, 21 and 22). When balanced, RT are associated with normal phenotypes. RT are the most common structural chromosomal abnormalities observed in humans, with an incidence of 0.123%, the most frequent being the t(13;14) with an incidence of 0.097% (Jacobs, 1972
; Nielsen and Wohlert, 1991). RT carriers have a greater chance of being infertile, producing offspring with chromosomal abnormalities, or having multiple spontaneous abortions (Munné et al., 1998). Very little is known about the extent and the timing of selection against the embryos that are carriers of unbalanced translocations. Previous studies in animals revealed the selective elimination of embryos with unbalanced chromosomal translocations at early cleavage stages (Sonta et al., 1984). In humans, the possibility of selecting normal embryos by in-vitro culture until blastocyst was suggested (Ménézo et al., 1997, 2001), but no evidence of selection was demonstrated when day 3 embryos, from carriers of various translocations, were analysed from one blastomere and then cultured until day 5 (Evsikov et al., 2000). We present here our results on the developmental potential of human embryos from carriers of RT t(13;14), analysed by FISH with locus-specific probes. The analysis was carried out 3 days after insemination, performed by ICSI, on one or two biopsied blastomeres, and/or on day 5 spare embryos reanalysed for confirmation of diagnosis. |
Materials and methods |
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Patients
Patient characteristics and PGD cycles are described in Table I
. Five couples underwent nine PGD cycles for RT t(13;14). Couple 2 previously experienced six spontaneous miscarriages in natural cycles. Couple 4 underwent 10 previous IVF attempts without PGD. Couple 1 underwent two previous IVF cycles without PGD. No pregnancies were obtained in either case.
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Spare embryos were donated to science from couples after informed consent. The study was approved by the Ethical Board of the Erasmus Hospital with protocol number 99/121.
Stimulation protocol and oocyte recovery
Ovarian stimulation was performed using GnRH analogue (buserelin acetate: Suprefact spray; Hoechst Inc. Frankfurt, Germany), hMG (Humegon; Organon Inc. Oss, The Netherlands) and hCG (Pregnyl; Organon, Inc.). Oocyte retrieval was performed through vaginal puncture under ultrasound guidance. In-vitro oocyte culture and preparation for ICSI have been described elsewhere (Emiliani et al., 1999). Fertilization was performed in all cases by the ICSI technique.
Blastomere biopsy
One or two blastomeres were biopsied from 6–8-cell embryos at day 3, after having drilled a hole in the zona pellucida by laser (Fertilase; MTM, Switzerland).
FISH analysis
Biopsied blastomeres were spread on slides by the HCl–Tween 20 method, as described elsewhere (Coonen et al., 1994) and were then hybridized with a probe mixture containing a locus-specific probe (LS13q14), Spectrum Green (Vysis, Inc., Downers Grove, IL, USA), for chromosome 13, a telomeric probe (Tel 14q14), Spectrum Red, for chromosome 14 (Vysis, Inc.) and a centromeric probe (CEP 18), Spectrum Green + Spectrum Red, for chromosome 18 (Vysis, Inc.) for ploidy control. By this approach the embryos carrying normal or balanced chromosomes (displaying even spots for each probe) can be differentiated from embryos carrying unbalanced chromosomes (displaying uneven spots for one or two probes) (Scriven et al., 1998). Embryos were cultured until day 5 in ‘in-house made’ sequential media. The replacement of healthy embryos was performed on day 5, at the morula/blastocyst stage. Spare embryos were spread on slides in parallel on day 5, following the protocol mentioned above (Coonen et al., 1994) and were hybridized with the same probe mixture.
Embryo chromosome pattern classification
Chromosome patterns were classified according to criteria previously proposed (Delhanty et al., 1997; Sandalinas et al., 2001), as follows. Normal: 
90% of diploid cells; abnormal: 90% of uniformly abnormal cells; mosaic: (i) diploid or moderate mosaic: <90% and 62% of diploid nuclei with few abnormal nuclei, (ii) abnormal or extended mosaic: <62% of diploid cells for at least one chromosome analysed; chaotic (C): all nuclei showing randomly different chromosome patterns.
Statistical analysis
Statistical analysis was performed by 2-test, Mann–Whitney test and Kruskall–Wallis test: P < 0.05 was considered significant.
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Clinical results
A total of 132 oocytes was collected and 117 were inseminated by ICSI technique. Ninety-seven were fertilized (83%) and 65 of these, that reached 6–8-cell stages 72 h post-insemination, were biopsied and analysed in day 3, while four of them were analysed on day 5. In couple 3/cycle 1 only unbalanced embryos were found and in couple 5/cycle 2 the single embryo with a normal/balanced arrangement was blocked at the 5-cell stage. In both cases embryo replacement was not performed. Three clinical pregnancies were achieved in seven cycles with replacement, one which resulted in a spontaneous miscarriage (couple 5/cycle 3) caused by chromosome 22 trisomy and two that ended up in a delivery (patient 2/cycle 2 and patient 4/cycle 2) (Table I
). A fourth pregnancy was ectopic (couple 5/cycle 1). A total of twelve embryos was replaced and four sacs with fetal heart beat were observed in three patients (2, 4 and 5) (two singletons and one twin pair), 28 days after the embryo replacement. Amniocentesis was performed. In patient 4, the fetus was found to carry the balanced translocation. A twin pregnancy was obtained in patient 2. One of the twins has a normal karyotype and the second one is a carrier of the balanced translocation. Three babies were born.
Embryo FISH analysis
The FISH results are set out in Table II. In 19 embryos only one blastomere was analysed but in 15 of them reanalysis was performed on day 5. In 46 embryos PGD was performed on two blastomeres. For 48 embryos both PGD on day 3 and reanalysis on day 5 were performed. In six embryos reanalysis was unsuccessful, while PGD was unsuccessful in four further embryos, thus only day 5 analysis was performed. Twenty unbalanced embryos resulted from adjacent chromosome segregation, seven resulted from a 3:0 segregation, seven resulted from more complex chromosome rearrangement and two were polyploid. The proportions of unbalanced embryos (abnormal and abnormal mosaics) at different developmental stages are shown in Figure 1. The percentages of abnormal and abnormal mosaic embryos in the population of blocked embryos or blastocysts (80 versus 44% respectively) were significantly different (2: P < 0.05). All the embryos analysed on day 5 displayed a mosaic chromosome constitution. The coexistence of up to 19 different cell lines in the same embryo was observed; the principal diploid line was associated with a proportion of tetraploid cells and with further completely chaotic chromosome arrangements. In Table II the chromosome constitutions of each embryo are compared with their morphological stage. In Figure 2 the average percentages of diploid cells per embryo are shown for each chromosome analysed, at the three developmental stages. No significant differences were observed in the percentages of diploid cells per embryo for chromosome 14 at the three developmental stages (45.1 ± 22.9, 54.8 ± 22.2, 55.4 ± 30.1 in blocked embryos, morulae and blastocysts respectively), while the percentages of diploid cells per embryo for chromosomes 13 and 18 were significantly different between blocked embryos and blastocysts (chromosome 13: 43.1 ± 30.3*, 44.2 ± 30.9, 64.9 ± 29.0*; chromosome 18: 63.3 ± 23.2**, 74.4 ± 19.9, 83.0 ± 12.6** for blocked embryos, morulae and blastocysts respectively; Mann–Whitney test: P < 0.01). The mean percentage of diploid cells per embryos for all the embryos analysed was significantly higher for chromosome 18 than for 13 and 14 (chromosome 13: 49.1 ± 28.0*; chromosome 14: 53.0 ± 31.8**; chromosome 18: 75.7 ± 20.4***; Mann–Whitney test: P < 0.01). The average percentage of tetraploid cells per embryo was 17.5 ± 16.9, 9.7 ± 7.4 and 5.9 ± 8.9 in blocked embryos, morulae and blastocysts respectively. The differences between the three groups were not significant (Kruskall–Wallis test). Eight out of 24 blastocysts analysed on day 5 were diploid mosaics for the three chromosomes analysed, 12 were abnormal mosaics for one chromosome, two for two chromosomes, one for the three and only one embryo displayed 90% of diploid cells for the three chromosomes analysed and it was classed as normal. Two out of 12 morulae analysed on day 5 were diploid mosaics for the three chromosomes analysed, eight were abnormal mosaics for 1 chromosome, one for two and one was abnormal mosaic for the three chromosomes. Finally, two out of 15 embryos blocked on day 5 and analysed on the same day were diploid mosaics for the three chromosomes, four were abnormal mosaics for one chromosome, three for two chromosomes and six were abnormal mosaics for the three chromosomes analysed.
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Discussion |
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A high efficiency of PGD for RT t(13;14) was observed in our small series. Three out of seven cycles resulting in an embryo transfer led to a clinical pregnancy. The reliable selection of embryos that carry normal/balanced chromosomes by blastocyst culture, in cases of RT, was only observed in animals (Sonta et al., 1984
). In humans this issue remains a matter of controversy. The benefit of culturing embryos from translocation carriers up to blastocyst stage in IVF cycles without PGD was previously suggested from the high delivery rate observed (seven out of 11 cycles) (Ménézo et al., 1997, 2001). Conversely, in a larger study on 11 types of translocation, where the complete chromosome set of each embryo was analysed in one biopsied blastomere, no differences were observed in the percentages of normal, balanced or unbalanced embryos which reached the blastocyst stage (Evsikov et al., 2000). In our study, normal or diploid mosaic embryos developed better than abnormal mosaics. However, abnormal mosaic embryos represented 44% of blastocysts. It is of interest that all the embryos analysed on day 5, except one, were mosaics. A cut-off level of 10% of abnormal cells was fixed to discriminate real mosaic embryos from fully normal or fully abnormal embryos (Sandalinas et al., 2001) and a cut-off level of 38% of abnormal cells was fixed to discriminate between moderate or diploid mosaics and extended or abnormal mosaics (Sandalinas et al., 2001).
It is more difficult to classify the embryos when the proportion of tetraploid cells is considered. It is difficult to establish if tetraploid cell lines must be considered as normal lines in which cells are at the pre-mitotic stage, at the end of the S phase, or as abnormal cell lines. As previously stated, although polyploid cells in normal embryos are considered normal features of blastocyst formation in all mammalian species studied, a higher proportion of polyploid cells is probably detrimental (Ruangvutilert et al., 2000; Sandalinas et al., 2001). If we consider tetraploid cells as normal, six more blastocysts (3, 5, 8, 38, 41 and 43) must be reclassified as normal; one more morula (44) as normal; one more morula as diploid mosaic (26) and two more blocked embryos as diploid mosaics (15, 32). Thus a percentage of 70% of abnormal blocked embryos, of 61% of abnormal morulae and of 30% of abnormal blastocysts must be recalculated. Furthermore, if the proportion of tetraploid cells per embryo is added to the proportion of diploid cells, the percentages of normal cells per embryo for each chromosome are changed and they are, for blocked embryos: 60.2, 62.6 and 80.8%; for morula stage: 51.6, 62.2 and 81.8%; and for blastocysts: 70.8, 61.3 and 88.9% for chromosomes 13, 14 and 18 respectively. It is worthy of note that the differences in percentages of diploid/tetraploid cells between the three developmental stages, for the three chromosomes analysed, are less pronounced than in the case in which only a diploid population is considered. However, the degree of mosaicism was higher for the chromosomes implicated in the translocation. A higher degree of mosaicism for the chromosomes involved in a translocation, as compared to control chromosomes, was previously observed (Iwarsson et al., 2000). A higher tendency of acrocentric chromosomes (such as 13 and 14) to malsegregate during mitosis/meiosis or a predisposition to malsegregate from the translocation itself have been suggested as possible mechanisms (Iwarsson et al., 2000). These theories seem to be confirmed by our previous findings in which five chromosomes (18, X, Y, 21 and 13) of embryos from IVF couples with a normal karyotype were analysed and no differences were observed in the incidence of mosaicism for each chromosome (unpublished data). Furthermore, a differential selection level for each one of the three chromosomes analysed was observed in our study.
No significant variations in percentages of abnormal cells for chromosomes 14 were observed at the three developmental stages analysed, while a more pronounced reduction of abnormal cells for chromosomes 13 and 18 was detected, from blocked embryos up to blastocysts. These findings may indicate that different chromosomes can regulate early embryos in various ways. Consequently, care must be taken before coming to the general conclusion that the same selection mechanism applies to unbalanced arrangements of translocations involving different chromosomes. It remains very difficult to interpret the highly documented phenomenon of mosaicism, which seems to be an increasingly physiological feature of in-vitro cultured embryos (Delhanty et al., 1997; Conn et al., 1998; Evsikov and Verlinsky, 1998; Veiga et al., 1999; Emiliani et al., 2000; Ruangvutilert et al., 2000; Sandalinas et al., 2001; Bielanska et al., 2002). A mechanism of shunting of abnormal cells into the trophoblast, during the embryo development, was hypothesized but this theory is not supported by recently published data (Evsikov and Verlinsky, 1998; Magli et al., 2000) in which a high degree of mosaicism was observed even in the inner cell mass of blastocysts. It is probable that not all kinds of mosaicism have the same impact on embryo development and it is still not clear which is the correct cut-off level to discriminate between mosaic embryos that could or could not originate a normal fetus. In our case, embryos carrying an abnormal mosaic arrangement for one single chromosome more frequently reached the blastocyst stage. This indicates that it is not only the number of abnormal cells but also the number of chromosomes implicated in abnormal arrangements that are detrimental. What seems to be clear from our study is that in the case of RT t(13:14) the incidence of abnormal cells per embryo and of abnormal mosaic embryos is higher for the chromosomes implicated in the translocation. Thus, more precautions must be taken in performing a PGD in the case of carriers of RT. Finally, our approach of combining one locus-specific probe with one telomeric probe proved to be efficient and less labour intensive than patient-specific probes, as previously stated (Scriven et al., 1998; Munné et al., 1998; Conn et al., 1999; Escudero et al., 2000; Iwarsson et al., 2000).
In conclusion, the lower percentage of abnormal embryos and the lower number of involved chromosomes in abnormalities in blastocysts with respect to blocked embryos, indicate that normal embryos developed better. At the same time the 44% of abnormal blastocysts confirms that in-vitro culture up to day 5 is not sufficient to guarantee a complete selection against abnormalities. PGD remains the only efficient tool to guarantee the selection of a normal/balanced embryo. Finally, because of the high degree of mosaicism reported for the chromosomes implicated in the RT t(13:14), PGD based on FISH analysis of two cells is strongly recommended for this anomaly.
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This work was financially supported by the ‘Fond National de la Recherche Scientifique Medicale’, Convention No. 3.4602.00 and from the Erasme Foundation.
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4 To whom correspondence should be addressed. E-mail: semilian{at}ulb.ac.be
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References |
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Submitted on October 12, 2001; resubmitted on April 4, 2002; accepted on June 17, 2002.
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