1 UCL Centre for Preimplantation Genetic Diagnosis, Department of Obstetrics and Gynaecology, University College London, 8696 Chenies Mews, London, WC1E 6HX and 2 The London Fertility Centre, Cozen's House, 112a Harley Street, London W1G 7JH, UK
3 To whom correspondence should be addressed. Email: joyce.harper{at}ucl.ac.uk
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Abstract |
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Key words: aneuploidy mechanisms/blastocyst/chromosomal mosaicism/FISH/human embryos
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Introduction |
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Chromosomal analysis of embryos using several techniques including karyotyping, fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) has demonstrated frequent mosaicism that is largely independent of maternal age. Mosaicism mostly arises during cleavage and could have a major effect on embryonic survival. Karyotyping has revealed significant levels of mosaic aneuploidy (Papadopoulos et al., 1989; Jamieson et al., 1994
; Clouston et al., 1997
, Clouston et al., 2002
). However, technical difficulties in arresting the chromosomes in metaphase will always result in an underestimate of mosaicism in these studies. The application of single cell CGH provides the opportunity to assess the copy number of all the chromosomes in every cell of an embryo and thus identify the true level of mosaicism (Voullaire et al., 2000
; Wells and Delhanty, 2000
). In these two small series (24 embryos in all), 75% of embryos had at least one abnormal cell, but the labour-intensive nature of single cell CGH analysis precludes its widespread application. FISH has allowed chromosome enumeration to be performed on all interphase nuclei within an embryo without the need for culturing cells or preparing metaphase spreads. Thus, the FISH technique overcomes the problems that have arisen during karyotypic analysis and it has been possible to investigate the frequency, stage of onset and the mechanisms leading to chromosomal mosaicism in large groups of embryos. Different types of mosaicism have been reported after FISH analysis including aneuploid mosaics, polyploid and haploid mosaics, chaotic mosaics and multinucleation in cleavage stage embryos (Delhanty et al., 1993
, 1997
; Munné et al., 1994
; Harper et al., 1995
; Laverge et al., 1997
; Conn et al., 1998
; Munné and Cohen, 1998
; Gianaroli et al., 1999
; Iwarsson et al., 1999
, 2000
) as well as blastocysts (Evsikov and Verlinsky, 1998
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000a
).
Aneuploid mosaicism is considered to be the most frequent form of mosaicism originating in the first few embryonic divisions (Munné et al., 1994) and it is thought that it arises and persists due to the lack of expression of certain cell cycle checkpoint genes during the early cleavage stage (Delhanty and Handyside, 1995
). Aneuploid mosaics have been shown to arise due to mitotic non-disjunction (which causes reciprocal chromosome loss or gain in daughter cells); in addition, chromosome duplication and anaphase lag are thought to be likely mechanisms (Delhanty et al., 1997
). Ploidy mosaics have also been reported frequently (Harper et al., 1995
; Clouston et al., 1997
; Delhanty et al., 1997
; Munné et al., 1997
; Staessen et al., 1999
), with tetraploidy predominating and more rarely haploidy and triploidy. It has been proposed that tetraploid cells may be a normal feature of the development of the trophectoderm (Drury et al., 1998
). Tetraploid trophectoderm cells may arise as a result of endoreduplication or endomitosis and possibly play a role in embryo implantation (Drury et al., 1998
). The origin of diploid/haploid and diploid/triploid mosaicism is not clear and more difficult to explain. Chaotic mosaic embryos have been reported in many studies and are defined as embryos where all or a majority of nuclei show a randomly different chromosome complement. They have been reported in cleavage stage embryos (Harper et al., 1995
) as well as at the blastocyst stage (Evsikov and Verlinsky, 1998
).
When using FISH to analyse chromosome constitutions, several technical obstacles emerge, including failure of hybridization, signal overlapping yielding false-negative results, and split or diffused signals (Munné et al., 1998; Ruangvutilert et al., 2000a,b
). FISH is also limited by the number of probes that can be applied simultaneously due to an increasing risk of FISH artefacts and hybridization failure with increasing numbers of probes (Ruangvutilert et al., 2000b
). These factors have led to an underestimate of chromosome loss as a cause of mosaicism. The use of differentially labelled probes at two different loci to detect a single chromosome has been shown to increase accuracy of detection by reducing scoring errors and has confirmed that low level mosaicism is not a FISH artefact owing to hybridization failure or overlapping signals (Conn et al., 1999
; Magli et al., 2001
).
To avoid these problems, in this study, chromosomes 1, 11, 18, X and Y of day 5 embryos were analysed using three sequential rounds of FISH with two probes for each of the autosomes. The aims were to determine the true level of mosaicism by excluding FISH artefacts and thereby obtain information concerning the mechanisms responsible for generating aneuploidy mosaicism.
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Materials and methods |
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Superovulation
Pituitary downregulation was achieved by the administration of GnRH analogue depot injection (Prostap, leuprorelin acetate; Wyeth Laboratories, UK) 3.75 mg or nasal spray (Synarel, nafarelin acetate; Pharmacia, UK) 2 mg/ml per day starting on cycle day 2 for 1013 days. Ovarian stimulation was commenced using recombinant FSH (Puregon; Organon, UK) either 150 IU/day if aged <38 years or 225 IU/day if 38 years. The patients were monitored with serum estradiol levels and vaginal ultrasound regularly starting on day 6 of stimulation, and the dose of FSH was adjusted accordingly. Between days 12 and 14 of stimulation, patients who had a cohort of follicles of 1820 mm in diameter were given 10 000 IU of HCG (Pregnyl; Organon, UK) to trigger ovulation. Thirty-six hours later, follicular aspiration was carried out under transvaginal ultrasound guidance.
Embryo culture
Oocytes were retrieved using flushing medium [supplemented with sodium pyruvate, human serum albumin (HSA, heparin 10 IU/ml, penicillin 50 000 IU/l, streptomycin 50 mg/l and HEPES; Medicult UK Ltd], incubated in 6% CO2 in air, at 37°C, inseminated and cultured in 500 µl of IVF medium (bicarbonate-buffered medium containing HSA, penicillin and sodium pyruvate; Vitrolife, Scandinavia). On day 1, oocytes were assessed for the number of pronuclei and transferred into 25 µl microdroplets of cleavage medium (bicarbonate-buffered medium containing HSA, penicillin-G, EDTA, glucose, inorganic salts and amino acids; G-1, Vitrolife, Scandinavia). On day 3, the best embryos were selected for transfer and suitable spare embryos were cryopreserved.
Embryo group categorization
The embryos were divided into two groups depending on the culture medium. Group I embryos were cultured in standard cleavage medium (6.1 Vitrolife, Scandinavia) from day 0 to day 5, and group II embryos were cultured in standard cleavage medium from day 0 to day 3 and then in blastocyst medium (6.2 Vitrolife, Scandinavia) from day 3 to day 5. Only embryos that arose from a bipronucleate zygote were included in the study.
Embryo spreading
Embryos were spread as described previously (Harper et al., 1994; Ruangvutilert et al., 2000a
). Each embryo was washed in phosphate-buffered saline (PBS) to remove excess culture medium. The embryo was transferred to a small drop of spreading solution (0.01 mol/l HCl, 0.1% Tween-20) on a poly-L-lysine-coated slide. The spreading solution was used to dissolve the zona pellucida and cytoplasm. Subsequently, the embryonic nuclei were washed by gentle agitation of the spreading solution using a polycarbonate capillary, until free of cytoplasm. The embryo was constantly observed under an inverted microscope. The slides were left to air dry, washed in PBS for 5 min and dehydrated through an ethanol series (70, 90 and 100%). All embryonic nuclei were mapped with the aid of an England Finder (Graticules Ltd, UK) under a phase-contrast microscope.
Control analysis
For each FISH procedure, a control slide from a normal male lymphocyte culture was used to assess the efficiency of the FISH by randomly scoring 100 interphase nuclei. Moreover, a set of interphase nuclei (1020) and metaphases (25) were mapped as representative results from each control slide, using an England Finder, and analysed in the same way as the embryos using three rounds of FISH.
FISH procedure
FISH was performed in three sequential rounds. The first and second rounds were performed with probes for chromosomes 1, 11 and 18, whilst the third round used probes for the sex chromosomes and chromosome 18 (Table I). All probes except one were obtained from Vysis (UK) Ltd. The first round included the following probes: 1p SpectrumGreen (telomere CEB108/T7), which hybridizes to the subtelomere region of the short arm of chromosome 1; 11q SpectrumOrange (telomere VIJyRM2072), which hybridizes to the subtelomere region of the long arm of chromosome 11; and CEP18 SpectrumAqua (-satellite D18Z1), which hybridizes to the centromere region of chromosome 18. The second round included the following probes: 1sat-III (satellite II/III), which hybridizes to the heterochromatic region of chromosome 1 (laboratory prepared); CEP11 SpectrumGreen (satellite D11Z1), which hybridizes to the centromere region of chromosome 11; and 18q SpectrumOrange (telomere VIJyRM2050), which hybridizes to the subtelomere region of the long arm of chromosome 18. The third round included the following probes: repeated use of CEP18 SpectrumAqua (as an internal control); CEPX SpectrumGreen (
-satellite DXZ1), which hybridizes to the centromere region of chromosome X; and CEPY SpectrumOrange (
-satellite DYZ3), which hybridizes to the centromere region of chromosome Y (Table I).
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After the analysis of the first round, all probes were washed off using 4x SSC/0.05% Tween-20 for 5 min (x2) and in PBS for 10 min at room temperature. Aliquots of 5 µl of the second hybridization solution were applied to the glass slide, covered with a 13 x 13 mm coverslip and co-denatured at 75°C for 5 min. The slides subsequently were left to hybridize at 37°C in a moist chamber overnight. The post-hybridization washes consisted of 3 x 10 min in 50% formamide/2x SSC, 1 x 10 min in 2x SSC and 1 x 5 min in 2x SSC/0.1% NP-40. The slides were air dried, mounted and analysed in the same way as in the first round. The embryonic nuclei were specifically identified with the aid of a map previously drawn using an England Finder.
Probes of the second round were washed off as described previously. The third round X/Y/18 probe mix was applied to the slides and co-denatured at 75°C for 5 min. The slides were incubated in a moist chamber at 37°C for 2 h and subsequently were washed, mounted and analysed as described previously.
Criteria for signal scoring
The criteria for signal scoring were as used by Hopman et al. (1988), which proposed that signals had to be a minimum of a signal's width apart to be scored as two separate signals.
Mosaicism and events
All embryonic chromosome patterns were classified using the same criteria as Delhanty et al. (1997) described for cleavage stage embryos.
Diploid mosaic embryos with aneuploid cells were considered to have arisen through three different mechanisms: (i) when the embryo contained cells with monosomies, then the mechanism was classed as chromosome loss (CL); (ii) when the embryo contained cells with trisomies, then the mechanism was classed as chromosome gain (CG); and (iii) when the embryo had monosomies and trisomies of the same chromosome(s) in different cells, this was classified as mitotic non-disjunction (MND). Nuclei with multiple abnormalities affecting at least three chromosomes were classed as chaotic and not included in the analysis of the events. This included nullisomies and tetrasomies.
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Results |
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Five arrested embryos were not included, since the signals were not analysable due to loss of most nuclei during sequential rounds of FISH and poor quality nuclei. Embryos from both groups included nuclei which showed contradictory information for the two probes used for each autosome in the different rounds of FISH. These cells were excluded from the analysis of events and classed as inconsistent results.
FISH analysis of controls
Each FISH experiment included a control male lymphocyte slide with mapped nuclei in order to assess the efficiency of probe hybridization in the sequential rounds. Overall, 87.1% (range 7896) of the control nuclei showed normal signals for all eight probes used. Subtelomeric probes for chromosomes 1p, 11q and 18q showed a higher incidence of one signal per chromosome per nucleus, 7.8 (range 411), 3.1 (range 1.24.5) and 3.9% (range 25), respectively. Heterochromatic region or centromeric probes, 1satII/III, 11CEP and 18CEP (in both the first and third rounds), displayed a lower incidence of one signal per chromosome per nucleus, 6.2 (range 4.47.5), 2.4 (range 0.93.3) and 2.2% (1.53.4), respectively. Nuclei with one signal for the X chromosome and no signal for chromosome Y and nuclei with three or more signals for autosomes comprised <1%. Furthermore, all three subtelomeric probes, 1p, 11q and 18q, showed the occurrence of split signals (i.e. replicated DNA) in 2.2, 1.4 and 1.9% of nuclei, respectively, which was considered normal due to the position of the probes.
FISH analysis of embryos
Twenty-one embryos were analysed for each group. FISH results for group I and group II embryos are shown in Tables II and III, respectively.
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Discussion |
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Prevalence and type of mosaicism
The opportunity also arose during the study to compare the effects of transferring some embryos on day 3 of culture from the standard cleavage medium to the more enriched blastocyst development medium (group II). Both groups showed a high prevalence of mosaicism; 95% (20 out of 21) of embryos were mosaic for group I and 90% (19 out of 21) of embryos were mosaic for group II. These findings are similar to the findings from other studies, which have analysed blastocysts either by karyotyping (Clouston et al., 1997, 2002
) or by FISH analysis (Evsikov and Verlinsky, 1998
; Veiga et al., 1999
; Magli et al., 2000
; Ruangvutilert et al., 2000a
). In the present study, the proportion of cells testing normal (diploid) per embryo amounted to 73% for group I and 78% for group II. These results are also comparable with those of other studies, which have shown a mean of 72% of all analysable nuclei per blastocyst normal for the tested chromosomes (Coonen et al., 2004
).
The predominant type of mosaicism in this study was that of diploid/aneuploid, affecting 32 of the 42 embryos. However, in 20 of these embryos, other cell lines were also present including polyploid, haploid and chaotic types. The available evidence suggests that aneuploid mosaicism detected at the cleavage stage is generated during one of the first three divisions, typically at the first or second division (Delhanty et al., 1997; Munné et al., 2002
; Katz-Jaffe et al., 2004
). Data from these day 5 embryos suggest that if the aneuploid cells arose during cleavage, they are at a selective disadvantage compared with the diploid cells which have outgrown them, since both groups were comprised of embryos which had grown beyond the cleavage stage. Alternatively, anomalies affecting a few cells or a single cell may have been generated in later divisions since for most embryonic genes, maximum expression does not occur until the blastocyst stage (D.Wells et al., in preparation). Reduced numbers of transcripts from certain of the cell cycle checkpoint genes could allow errors to arise during days 4 and 5. Although there are many single cell anomalies, in some cases the total number of aneuploid cells per embryo is significant and, bearing in mind that only a few chromosome pairs were tested, there are likely to be many more undetected abnormalities in these embryos. The general consensus is that embryos with less than half of their cells truly normal diploid would be unlikely to survive beyond the implantation stage (Munne et al., 2004).
Tetraploidy
The phenomenon of tetraploidy, especially in blastocyst studies, has been well documented. It is considered as a normal feature of human embryonic development (Evsikov and Verlinsky, 1998). In our study, there was a mean of 1.9 and 7% of tetraploid cells per embryo for group I and group II, respectively. The difference in frequency of tetraploid cells as a result of changed culture medium was not unexpected, but also raises the question of whether the development of tetraploid cells is favoured by certain culture conditions. It has been proposed that pure polyploid embryos or diploid/polyploid embryos might arise from failure of cytokinesis (Harper et al., 1995
; Munné and Cohen, 1998
) or from cell fusion (Benkhalifa et al., 1993). The existence in our study of a few triploid cells and cells of higher ploidy suggests that these may have been formed by cell fusion. It is of interest in this context that a recently established line of human trophectoderm cells exhibits syncytium formation but the nuclei remain diploid (H.Moore, personal communication).
Distinction between artefacts and true results
The use of two probes per autosome allowed this study to detect an error rate of 5% per nucleus and to exclude those artefacts from the analysed results. For both groups, a true chromosomal error in a total of 76 nuclei would have been missed if only one probe per chromosome had been used. Therefore, by adopting our chosen strategy, we were able to detect the true levels of mosaicism for the three autosomes studied. The probe that showed the highest rate of failure of hybridization on the embryonic material was the subtelomere probe for chromosome 18 (Table VIII). Subtelomeric probes generally have lower hybridization efficiency than probes that detect repeat sequences, but in this case the situation possibly was exacerbated because the probe was used in the second treatment round. However, in the lymphocyte control nuclei, the probes for chromosome 1, both for the subtelomeric (used in the first round) and the heterochromatic regions, showed the highest failure rates.
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Mechanisms of aneuploidy mosaicism
In the current study, CL was the predominant mechanism leading to mosaicism in both groups of embryos, being responsible for 50% of aneuploid cells in group I and 40.5% in group II. CG followed, with 44% in group I and 35.1% in group II. However, only 6% of aneuploid cells occurred due to MND in group I, while four times this percentage arose by this mechanism in group II The differential involvement of chromosomes in MND (predominantly chromosome X in group II which was not involved at all in group I) is interesting but may well be due to chance.
Similarly, it is of interest that in group I embryos, in which growth had slowed or arrested, chromosome 1 showed a high incidence of loss but in group II embryos, which had continued dividing, chromosome 1 was not affected by loss at all. This finding is in accordance with findings of Sandalinas et al. (2001) which revealed that embryos fully monosomic for the larger chromosomes fail to reach the blastocyst stage. This might possibly be due to the fact that the presence of cells with monosomies of such a large chromosome would have a detrimental effect on development and had been selected against in the more rapidly dividing group II embryos. Trisomy 18 caused by CG was seldom found in either group, which is a similar finding to that reported recently by Coonen et al. (2004)
. Furthermore, the occurrence of monosomy 18 was shown to be high (especially in group II embryos), indicating that chromosome 18 might be more prone to CL compared with the other autosomes and gonosomes tested.
The current study shows that CL and CG are the mechanisms mostly affecting human day 5 embryos. CL is the predominant mechanism in this study leading to mosaicism; furthermore, analysis of a larger pool of embryos may prove such a finding statistically. This reinforces data on day 3 embryos obtained earlier by our group using dual locus-specific yeast artificial chromosome (YAC) and plasmid probe combinations for various autosomes (C.Conn and J.D.A.Delhanty, unpublished observations). Chromosome loss is presumed to occur via anaphase lag, in this case during mitosis. Coonen et al. (2004) concluded in their study on a much larger number of blastocysts that anaphase lagging is the major cause of chromosomal mosaicism. However, since they were using a single probe for each chromosome, they were only able to count as valid those abnormalities affecting at least two cells. In relation to the findings in these two studies, it is of considerable interest that aneuploidy screening of cleavage stage embryos has shown that CL is more common than CG as a cause of constitutional aneuploidy arising during meiosis (Munné et al., 2004
).
During this study, the average maternal age was 34 years but there was still a high prevalence of mosaicism. The three embryos that were uniformly diploid for the tested chromosomes were donated by three women <30 years of age, whereas six women whose age was >38 years donated nine embryos, which were either diploid/aneuploid mosaic or diploid/aneuploid/chaotic mosaic. While these observations may indicate a trend, clearly the numbers in our study are too small to enable analysis of a maternal age effect. However, a report of a study of a large number of cleavage stage embryos from women in an older age group showed a significant association of mitotic aneuploidy with advanced maternal age (Munné et al., 2002). The association was particularly marked for the MND category. In that study, CL was only a third as frequent as MND and consequently mitotic anaphase lag failed to show a significant increase with maternal age. The increased frequency of MND may be due to the age of the group studied but, since only a single probe per chromosome was used, a cut off point of 10% was used to avoid FISH error, i.e. embryos with <10% abnormal cells were considered normal, almost certainly leading to an underestimate of anomalies affecting a single cell. Possibly a study of a large number of embryos using two probes per chromosome might show a significant association of mitotic anaphase lag with increasing maternal age.
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Acknowledgements |
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References |
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Submitted on May 13, 2004; accepted on September 17, 2004.