1 Departments of Obstetrics and Gynecology, 2 Human Genetics and 3 Experimental Medicine, Royal Victoria Hospital, McGill University, Montreal, QC H3A 1A1, Canada
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Abstract |
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Key words: chromosomes/embryo mosaicism/fluorescence in-situ hybridization/preimplantation development
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Introduction |
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Despite the prevalence of mosaicism in cleavage stage embryos, little is known about the effect of this condition on embryonic development and pregnancy outcome. Mosaicism has been reported in both fetal and placental tissues of first and second trimester pregnancies, but its incidence at these stages is very low. It is present in only ~5% of aneuploid spontaneous abortions at 620 weeks gestation (Hassold, 1982) and in 12% of viable pregnancies screened by chorionic villus sampling (CVS) (Ledbetter et al., 1992
; Wang et al., 1993
). Furthermore, unlike in embryos, the mosaicism detected in established pregnancies is mostly limited to diploid trisomy. The considerable reduction in mosaicism from early cleavage stages to clinically established pregnancies indicates that most mosaicism is eliminated prior to the first trimester. Cytogenetic analysis of blastomeres from embryos on day 2 to day 5 of development in vitro suggests a progressive loss of chromosomally abnormal embryos during preimplantation development (Almeida and Bolton, 1996
). Several studies reported that the incidence of chromosomal anomalies at early cleavage divisions is higher among arrested compared with non-arrested embryos (Munné et al., 1994a
, 1995a
,b
; Bahce et al., 1999
). It is therefore possible that the selection against mosaic embryos may begin during preimplantation development. This early wastage of embryos may be a contributing factor to the low rates of blastocyst formation and high rates of implantation failure following embryo transfer on days 23 post-insemination.
In order to help clarify the significance of co-existing diploid and non-diploid cells for preimplantation embryo wastage, implantation failure, and fetal and placental mosaicism, we (i) used multi-probe FISH to determine the incidence of mosaicism in 2-cell to blastocyst stage embryos, (ii) compared the composition (type and numbers of non-diploid cells) of mosaicism detected at each stage, and (iii) examined the relationship between the different patterns of mosaicism and embryonic development and viability.
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Materials and methods |
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DNA probes
Directly labelled DNA probes were obtained from Vysis (Downers Grove, IL, USA). The probes varied according to their region of hybridization on the chromosome and fluorescent label as follows: CEP 2 (alpha satellite DNA, spectrum orange); CEP 7 (alpha satellite D7Z1, spectrum green); LSI 13 (region 13q14, spectrum red); CEP 16 (satellite II, D16Z3, spectrum aqua); CEP 18 (alpha satellite D18Z1, spectrum aqua, or spectrum blue); LSI 21 (region 21q22.1321q22.2, spectrum green); LSI 22 (region 22q.11.2, spectrum gold); CEP X (alpha satellite DXZ1, spectrum green); CEP Y (alpha satellite DYZ3, spectrum orange). Each of the probes had been previously tested in our laboratory by hybridization to 200 normal 46,XY lymphocytes prepared by standard cytogenetic procedures (not reported here). The hybridization efficiency of each probe was determined as the number of interphase cells with the expected number of probe-specific signals, minus cells with less than or more than the expected number of signals divided by total number of cells scored. The efficiencies for the probes varied from 89 to 98%.
FISH protocol
Embryos were washed in phosphate-buffered saline (PBS) and transferred onto poly-L-lysine-coated slides. Cleavage stage and blastocyst stage embryos with intact zona pellucida were dissolved in 0.01 N HCl, 0.1% Tween 20 solution and nuclei spread and fixed on the slides (Coonen et al., 1994). The preparations were air-dried, washed in PBS for 5 min and dehydrated in 50, 70 and 100% ethanol. Fixed nuclei were viewed using a phase contrast microscope and their location marked with a diamond pen. Embryonic nuclei were pretreated with pepsin (0.1 mg/ml in 0.01 N HCl) for 10 min at 37°C, and again dehydrated. FISH probes for three to five different chromosomes were combined in hybridization buffer [50% formamide, 2xsaline sodium citrate (SSC); Oncor, Gaithersburg, MD, USA]. Probe mixtures used in the study included a three-colour probe mixture specific to chromosomes 2, 7, 18, a three-colour mixture specific to chromosomes X, Y, 18, and a five-colour mixture probe mixture specific for chromosomes 13, 16, 18, 21, 22. The probe mixtures were applied to the embryonic nuclei and sealed under a plastic coverslip. The target and probe DNA were codenatured by heating the slide to 7478°C, for 46 min. Hybridization was carried out in a moist chamber at 37°C, for 518 h. The unbound probe was washed off using 0.4xSSC/0.3% Tween 20, or 0.7xSSC/0.1% Tween 20 solution at 73°C, for 26 min, followed by rinsing in 2xSSC/0.1% Tween 20 at room temperature. Preparations were mounted in antifade solution (p-phenylenediamide dihydrochloride in PBS; Vector). Depending on fluorescence colour of probes used, antifade was applied alone, or with DAPI (Sigma) nuclear stain (0.250.5 ng/ml). Nuclei were viewed using a fluorescence Olympus DX 60 microscope equipped with appropriate filters. Images were captured using a CCD camera and Cytovision software (Applied Imaging, Santa Clara, CA, USA).
Signal and data analysis
Only intact, undamaged nuclei were scored. Nuclei with a dim signal for more than one of the chromosomes tested were considered a hybridization failure and were not scored. Fluorescence spots of diameter noticeably smaller than other signals in the same nucleus were considered non-specific probe binding and were excluded from the results. Two small focal signals of the same colour, separated by a distance of less than one signal domain, were considered to represent a split signal from one chromosome. An embryo was diagnosed with an imbalance for a specific chromosome when the abnormal signal pattern was detected in a higher proportion of cells than in normal control lymphocytes. Polyploidy (more than two signals for each chromosome pair tested), haploidy (a single signal for each chromosome pair tested), or reciprocal gains and losses in sibling blastomeres were always considered as true imbalances. Only those embryos yielding results in 75% of cells were included in the study.
The incidence of mosaicism and the percentage of normal to abnormal cells in mosaic developing and arrested embryos were compared at sequential stages of preimplantation development (2-cell to blastocyst stages). Mosaicism was compared among cleavage stage embryos of different morphological grades. The frequency of mosaicism in cleavage stage embryos was also compared among three different maternal age groups (34, 3539,
40 years of age). Statistical analysis was carried out using
2-test and Fisher's exact (F) test.
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Results |
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FISH results of 216 embryos are presented in Table I. Overall 29.6% of embryos were uniformly normal (diploid; 2N) for the chromosomes tested. Twenty-two per cent of embryos comprised only abnormal cells. Nine per cent of embryos were chaotic, with blastomeres showing various imbalances for multiple chromosomes. Seven per cent were aneuploid. One of these embryos showed double uniform aneuploidy, trisomy 13 and monosomy 21. One aneuploid embryo showed trisomy 2 in all 11 cells plus chaotic imbalances for chromosomes 7 and 18 in two of the cells. Another embryo showed trisomy 18 in 7/8 cells and chaotic complement in one cell. Haploidy and polyploidy accounted for 0.5 and 5.6% of embryos respectively. The largest cohort of embryos (48.1%) was mosaic, consisting of combinations of normal and abnormal cells. These embryos were classified into four groups based on the predominant type of non-diploid cells detected (Table I
). Sixteen per cent of embryos contained both diploid and chaotic cells. This was followed by 2N-polyploid mosaicism, consisting predominantly of tetraploid (4N) cells (14.8%) and 2N-aneuploid mosaicism consisting of diploid cells and cells showing a gain and/or a loss for one of the chromosomes tested (14.4%). Only 2.8% of the embryos contained combinations of diploid and haploid (1N) cells. The proportion of haploid cells in the one 2N-haploid blastocyst was 55%.
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The incidence of mosaicism was compared at each developmental stage. The overall frequency of mosaicism increased significantly at sequential stages of preimplantation development from 15.2% at the 24-cell, to 49.4% at the 58-cell, to 58.1% at the morula and to 90.9% at the blastocyst stage (2-test, P < 0.0001; Table II A
). The increase in frequency of overall mosaicism was found in both developing and arrested embryos (Table II B and C
). The majority of the non-diploid blastomeres observed in the 2-cell to morula stage mosaic embryos were chaotic or aneuploid. Chaotic mosaicism decreased significantly from the arrested morula stage to the blastocyst stage embryos (F-test, P < 0.04). Diploid-aneuploid mosaicism also decreased by the blastocyst stage; however, because blastocysts were analysed for only three chromosomes, the rate of mosaic aneuploidy at the blastocyst stage may be slightly higher than the rate detected. The predominant form of mosaicism found at the blastocyst stage was diploid-polyploid (mostly tetraploid) mosaicism. The increase in 2N-polyploid mosaicism from both developing and arrested morulae to the blastocyst stage was statistically significant (F-tests, P < 0.0001).
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Discussion |
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Our FISH results revealed four major types of diploid mosaicism. One of the two most frequent types afflicting 2-cell to morula stage embryos comprised diploid cells and cells showing `chaotic' imbalances, often involving all of the chromosomes tested. These imbalances may have originated in a normal zygote or may have been superimposed on chromosome-specific meiotic or mitotic gains or losses and may be a consequence of chromosome misalignment on a disorganized spindle, in combination with a non-functional metaphase/anaphase checkpoint control (LeMarie-Adkins et al., 1997; Harrison et al., 2000
). The abnormalities of the mitotic spindle could be related to suboptimal in-vitro culture environment (Pickering et al., 1990
; A'arabi et al., 1997
).
As observed previously (Munné et al., 1994a,b
, 1995b
; Magli et al., 2000
), chaotic chromosome complements in this study were more frequent among arrested embryos. Detection of chaotic mosaicism up to the 12-cell stage demonstrates that blastomeres with extensive chromosome imbalances may not always compromise the first four cleavage divisions. This may be explained by the relatively late activation of the human embryonic genome at the 48-cell stage (Braude et al., 1988
). However, the majority (8/10) of morulae with chaotic cells were arrested. The two which were not arrested had only a low proportion of abnormal cells. In agreement with previous findings, the incidence of blastocyst chaotic mosaicism was significantly reduced (Evsikov and Verlinsky, 1998
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000
). Together, these data suggest that most extensive imbalances are incompatible with blastocyst formation. Early wastage of chaotic embryos is consistent with the rarity of multiple chromosome anomalies, and the very low rates of double trisomies (0.212.8% for all chromosomes combined) among spontaneous abortions (Reddy, 1997
). Replacing embryos reaching the blastocyst stage has yielded higher rates of implantation per embryo transferred compared with day 2 or 3 embryo transfer (Edwards and Beard, 1999
; Coskun et al., 2000
). Our study suggests that the greater implantation success following replacement of embryos cultured to the blastocyst stage in vitro may be associated with lower rates of the chaotic non-viable abnormalities among these embryos.
The second most frequent form of mosaicism detected in cleavage stage embryos in this study consisted of 2N cells in combination with cells showing imbalances for one of the chromosomes tested (aneuploid mosaicism). Diploid and trisomic and/or monosomic cell lines may originate from chromosome-specific mitotic non-disjunction, chromosome duplication or chromosome loss in selected cells from a diploid or an aneuploid zygote (Ford et al., 1988; Robinson et al., 1995
; Los et al., 1998
). The predominance of the diploid cell line among the aneuploid mosaic embryos, as well as the lack of a higher frequency of mosaicism in embryo donors aged
40 years, suggests that most of these cases originated from a mitotic error in a diploid zygote.
Recently, it was demonstrated that aneuploid cell lines may persist to the blastocyst stage from day 3 post insemination (Magli et al., 2000). However, relative to cleavage stage embryos, the number of aneuploid cells in the five 2N-aneuploid blastocysts examined in this study were low (68 cells). Relatively small numbers of aneuploid cells have also been detected in other blastocysts (Evsikov and Verlinsky, 1998
; Ruangvutilert et al., 2000
). The minor aneuploid cell lines indicate that the aneuploid cells did not persist from early cleavage, but were formed at, or shortly before, blastulation, and suggest that most cleavage stage embryos with a high degree of mosaic aneuploidy do not complete preimplantation development.
Unlike that which has been observed in tetraploid-diploid mouse aggregation chimeras (James and West, 1994), recent studies of human blastocysts did not show evidence of preferential allocation of aneuploid cells into the trophectoderm cell lineage (Evsikov and Verlinsky, 1998
; Magli et al., 2000
). Therefore the small numbers of aneuploid cells detected in our cohort of blastocysts may have originated from the ICM or from trophectoderm, and could lead to generalized or to confined placental mosaicism (CPM) (Kalousek and Dill, 1983
).
A diploid-haploid (2N/1N) chromosome complement comprised the least frequent class of mosaicism detected in this study. The origins of haploid cells in diploid embryos have been discussed previously (Bongso et al., 1991; Delhanty et al., 1997
). In most cases in this, and other, studies, haploidy was limited to one or two blastomeres per embryo suggesting that 1N cells have a proliferative disadvantage among 2N cells (Munné et al., 1994a
, 1994b
; Harper et al., 1995
; Bahce et al., 1999
). However, our finding of one blastocyst with a large fraction (55%) of haploid cells demonstrates that haploid cells may occasionally proliferate throughout the preimplantation period. As 2N/1N mosaicism has not been documented in fetal tissues, such embryos must become eliminated at, or shortly after, implantation.
A small percentage of 28-cell embryos, ~2% of morulae, and ~89% of the blastocysts stage embryos in this study showed 2N-polyploid mosaicism. This form of mosaicism was also common in human morula and blastocyst stage embryos examined by others (Angell et al., 1987; Benkhalifa et al., 1993
; Clouston et al., 1997
; Evsikov and Verlinsky, 1998
; Ruangvutilert et al., 2000
). Polyploid cells have also been observed in a normal first trimester human placenta (Sarto et al., 1982
). The dramatic increase in incidence of polyploid cells from day 4 to day 6 of development in this study suggests that most polyploid cells arose during blastocyst formation, and may, as in other mammalian species, be a hallmark of trophoblast differentiation (Barlow and Sherman, 1972
; Hare et al., 1980
; Long and Williams, 1982
; Murray et al., 1986
). However, the proportion of polyploid cells in five of the blastocysts was very high (3347%). If tetraploidy in these blastocysts represents impaired cytokinesis caused by suboptimal culture conditions, the blastocysts may be arrested and unable to implant. Alternatively, the 4N cells may continue proliferation and contribute to the 2% of cases of spontanous abortions (Eiben et al., 1990
).
A number of studies have shown that dysmorphic embryos exhibit a higher incidence of chromosomal abnormalities (Munné et al., 1994b, 1995b
; Almeida and Bolton, 1996
; Delhanty et al., 1997
). Degree of fragmentation and relative size of blastomeres did not predict the incidence of mosaicism among embryos in this study (Table IV
). The 38.2% mosaicism rate among grade II cleavage stage embryos in this study suggests that inclusion of embryos with even-sized blastomeres and a small degree (<10%) of fragmentation among cohort being transferred to the patient may result in replacement of embryos with aneuploid or chaotic cells.
In conclusion, this study demonstrates that post-meiotic errors leading to mosaicism may occur, and persist throughout preimplantation development in vitro. Our results suggest that the developmental potential of mosaic embryos is dependent on the type and proportion of non-diploid cells. Mosaicism involving multiple chromosomal anomalies, or a non-diploid karyotype in >50% of blastomeres per embryo, appears to reduce embryonic capacity to develop into a blastocyst. This form of mosaicism may contribute to the high rates of implantation failures following cleavage stage embryo transfer. In contrast, small numbers of aneuploid cells in early embryos may not be detrimental to successive cleavage divisions. If compatible with implantation, embryos with mild aneuploidy may be a candidate for fetal, or for confined placental mosaicism. Most mosaic polyploidy in the blastocysts appears distinct from the mosaicism observed at the early stages of cleavage division, and may be a normal part of mammalian trophoblast differentiation. The extent to which the different classes of mosaicism observed in the present study exist among embryos selected for transfer to patients and in preimplantation embryos in vivo, and their contribution to the low rate of fecundity in the human, remains to be investigated.
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Acknowledgements |
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Notes |
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Submitted on November 17, 2000; resubmitted on June 11, 2001
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References |
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accepted on October 8, 2001.