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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Abstract |
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Key words: blastocyst/FISH/human embryo/preimplantation genetic diagnosis/Robertsonian translocation
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Introduction |
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Materials and methods |
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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 68-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 HClTween 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, MannWhitney test and KruskallWallis test: P < 0.05 was considered significant.
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Results |
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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; MannWhitney 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***; MannWhitney 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 (KruskallWallis 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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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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Acknowledgements |
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Notes |
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References |
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Submitted on October 12, 2001; resubmitted on April 4, 2002; accepted on June 17, 2002.