1 The London Bridge Fertility, Gynaecology and Genetics Centre, One St Thomas Street, London SE1 9RY, 2 CRUK Clinical Centre at Leeds, Division of Cancer Medicine Research, St James University Hospital, Leeds LS9 7TF, UK, 3 Iakentro Advanced Medical Centre, Thessaloniki, 542 50, Greece and 4 School of Biology, University of Leeds, Leeds LS2 9JT, UK
5 To whom correspondence should be addressed. Email: katerinachatzime{at}hotmail.com
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Abstract |
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Key words: cell cycle checkpoints/confocal laser scanning microscopy/human preimplantation embryo/mitotic spindle/nuclear abnormalities
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Introduction |
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Analysis of chromosomes in nuclei of single cells, biopsied from cleavage stage human embryos, by multicolour fluorescence in situ hybridization (FISH) for preimplantation genetic diagnosis (PGD), initially to identify the sex of embryos in couples at risk of X-linked disease (Griffin et al., 1993), and more recently, for aneuploidy screening (Lamb et al., 1997
; Tesarik et al., 2000
; Lewis et al., 2001
; Gianaroli et al., 2002
; Verlinsky et al., 2002
; Munné et al., 2003
), has provided a powerful tool for interphase molecular cytogenetics, albeit on a limited number of chromosomes determined by the availability of probes and different fluorochromes. Using this approach, numerous studies have confirmed the high incidence of aneuploidy in human gametes and embryos and the well-established increase associated with advanced maternal age (Munné et al., 1995a
, 2002
). In addition, however, subsequent multicolour FISH analysis of all the nuclei in biopsied embryos either from fertile patients, which had been rejected for transfer following PGD for X-linked disease, or in surplus embryos from infertile patients, has revealed that about a third of embryos are chromosomally mosaic (Delhanty et al., 1997
). In these mosaic embryos, some nuclei have the normal diploid number of the chromosomes analysed, indicating fertilization with eusomic gametes, but other nuclei can be haploid, polyploid (most commonly tetraploid) or aneuploid (Harper et al., 1995
; Munné et al., 1995a
,b
, 2003
). At the chromosomal level, therefore, human embryos can be classified as diploid, diploid/haploid/polyploid or diploid/aneuploid mosaics. Furthermore, some embryos from fertile patients appeared to have a random distribution of chromosomal abnormalities in a majority of their nuclei, with some evidence that this was patient specific, and these were classified as chaotic (Delhanty et al., 1997
).
These postzygotic nuclear and chromosomal abnormalities arising during cleavage, often involving chromosomal malsegregation, closely resemble the genetic instability observed in tumour cells, suggesting that cell cycle checkpoints may not operate at these early stages (Delhanty and Handyside, 1994). To investigate this, and to examine whether spindle abnormalities contribute to chromosome malsegregation, we have used laser confocal scanning fluorescence microscopy with combinations of a DNA fluorochrome to visualize nuclei and chromosomes and several antibodies specific for either spindle- or centrosome-associated proteins in human embryos either developing at the normal rate at cleavage to blastocyst stages or in arrested, mainly cleavage stage embryos. These antibodies included anti-
-tubulin to label spindle microtubules and anti-
-tubulin and anti-acetylated tubulin to label spindle poles.
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Materials and methods |
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Ovarian stimulation
A standard protocol was used to induce ovulation and control timing of oocyte retrieval. Pituitary down-regulation was achieved by the administration of GnRH analogues (Cetrotide; Serono; or Orgalutran; Organon, for the first 1014 days, followed by ovarian stimulation with pure FSH (Metrodin; Serono, UK) or FSH and LH (Altermon; IBSA) or recombinant FSH (Puregon; Organon or Gonal-F; Serono) for 1214 days. The patients were monitored by ultrasound regularly starting on day 6 of gonadotrophin administration, and between days 12 and 14, when adequate follicular development had been demonstrated (3 follicles of 17 mm in diameter), 10 000 IU of hCG (Profasi; Serono; or Pregnyl; Organon) was administered to trigger ovulation. Thirty-six hours after the hCG administration, transvaginal ultrasound-guided oocyte retrieval was performed.
Oocyte retrieval
Oocytes were retrieved by flushing ovarian follicles with IVF 20 (Vitrolife) incubated in 5% CO2 in air at 37°C and subsequently fertilized by conventional IVF or ICSI and cultured until the day of transfer (day 3) in G1.2 medium (Vitrolife). Embryos of patients requesting blastocyst transfer were further cultured in CCMTM medium (Vitrolife) until day 5. Spare embryos from day 3 or day 5 transfers were processed for immunofluorescence analysis.
Human embryo fixation, immunostaining and confocal imaging
Embryos were rapidly transferred from culture to ice-cold methanol (BDH) and fixed for 10 min. Following fixation they were briefly washed in Ca2+/Mg2+-free phosphate-buffered saline (PBS; Gibco BRL) containing 2% bovine serum albumin (BSA; Sigma) and transferred into 10 µl drops of the primary antibodies (1:1000 dilution in PBS/BSA) under mineral oil (Sigma) and incubated at 4°C for 1 h. Primary antibodies included: (i) a rat monoclonal antibody specific for -tubulin (Serotec); (ii) and (iii) mouse monoclonal antibodies specific for
-tubulin and acetylated tubulin (Sigma). The embryos were then washed twice in PBS/BSA before being transferred into 10 µl drops of the appropriate secondary antibodies (1:500 dilution in PBS/BSA) containing 1 ng/ml 4,6-diamidino-2-phenylidole (DAPI; Sigma). All secondary antibodies were highly cross-adsorbed Alexa Fluor 488 or 594 conjugates (Molecular Probes). Following 1 h incubation in the secondary antibodies the embryos were washed twice in PBS/BSA and mounted on slides (BDH) in Vectashield antifade medium (Vector Laboratories, USA) under a coverslip. The coverslips were then sealed with nail varnish. The embryos were examined and images captured using the Olympus BX61 fluorescence microscope and the Cytovysion software (Applied Imaging) and/or a Leica TCS-SP laser scanning confocal microscope. Confocal image analysis was typically accomplished by capturing a z-series stack of 1 µm thick sections encompassing the entire embryo. In multiply-stained embryos, images were acquired sequentially to avoid bleed-through artefacts using the 488 nm line of an argon laser to image Alexa 488, the 568 nm laser line of a Kr laser to image Alexa 594 and an argon-UV laser to visualize DAPI staining of DNA. A x25 or a x100 UV-corrected oil immersion lens was used depending on the size of the embryo being imaged.
Embryo classification
Embryos were divided into three groups depending on their development on the day of immunolabelling. Embryos on days 3 and 4 post-insemination with 4 and
10 cells respectively, and embryos at the compact morula or cavitating blastocyst stage on day 5, were assigned to the normally developing group. All embryos with fewer cells were assigned to the arrested group. Completely fragmented embryos with patchy DAPI staining were classified as degenerate.
Classification of interphase nuclear and spindle abnormalities
All interphase and metaphase stage nuclei were carefully examined and counted in each of the embryos. Abnormalities of interphase nuclei were classified according to the criteria set out in Table I. In all cases, including the identification of anucleate blastomeres, the cell boundaries were clearly visible because of background cytoplasmic labelling with either the anti-tubulin antibodies or DAPI. At the blastocyst stage, cell boundaries in the mural trophectoderm were also visible, allowing the identification of some abnormalities, particularly binucleate blastomeres.
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Results |
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Discussion |
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Nuclear abnormalities, including binucleate, anucleate and multinucleate blastomeres, were common at all stages, particularly in cleavage stage and arrested embryos (Figure 3; Table IV). In addition, micronuclei (possibly arising from chromosome loss) and/or apoptotic nuclei were observed in 2025% of normally developing and arrested embryos between days 3 and 5 (data not shown). However, labelling of nuclei for DNA strand breaks has demonstrated that apoptotic nuclei are only present following compaction in normally developing embryos (Hardy, 1999
).
Binucleate blastomeres are thought to arise through failure of cytokinesis during cleavage (Hardy et al., 1993b). In one arrested 2-cell embryo with an anucleate fragment, both of the blastomeres were tetranucleate, suggesting that cytokinesis had failed in two successive cleavage divisions (Figure 3a,b). Interestingly, in another arrested 4-cell embryo, there were two large binucleate blastomeres and two smaller anucleate blastomeres/cytoplasmic fragments, strongly suggesting that in this case, instead of failure of cytokinesis, the actin microfilaments of the contractile ring had been displaced to one end of the cell (Figure 3e,f). This may therefore represent one mechanism underlying the frequently observed phenomenon of cytoplasmic fragmentation, particularly if, in the absence of a nucleus, further cycles of aberrant cytokinesis continue, as occurs, for example, following enucleation of mouse zygotes (Petzoldt, 1990
).
In the pig, actin microfilament polymerization has been shown to be important for oocyte maturation and early embryo development (Wang et al., 2000a) and is affected by the culture conditions (Wang et al., 2000b
). Furthermore, in contrast to in vivo-derived embryos, those generated in vitro, by in vitro maturation of oocytes, fertilization and culture, were characterized by reduced perinuclear filamentous actin, binucleate and anucleate blastomeres and poor development to the blastocyst stage (Wang et al., 1999
). The parallels with human preimplantation development in vitro following IVF are striking, and raise the possibility that suboptimal culture conditions may be responsible for these similar effects on human embryo development. A comparative cytoskeletal analysis of human embryos cultured in a variety of culture media may therefore give a better insight into whether different culture conditions have an effect on spindle formation.
The majority of mitotic spindles examined in normally developing embryos had normal astral or fusiform-shaped poles and were bipolar (Table V; Figures 4a1, 4a6, 2a3Figures 4a1, 4a6, 2a3). However, several types of spindle abnormalities were observed which were more frequent at cleavage stages on days 3 and 4 and in arrested embryos and virtually absent in blastocysts by days 6 and 7. These abnormalities included abnormal shape, defined as a poorly organized spindle lacking well-defined poles, and/or one or more chromosomes separate from the spindle resulting presumably either from congression failure or anaphase lag (Figures 1a1 3, 3c,d). The latter could explain the frequent detection of chromosome loss in interphase nuclei from cleavage stage embryos following analysis by multicolour FISH with chromosome-specific probes. For example, sequential FISH analysis of nine chromosomes revealed multiple chromosome losses including several clones of cells missing one or both chromosomes of a particular pair in a small series of arrested cleavage stage embryos (Harrison et al., 2000). Of particular interest, however, is the observation of several tripolar (Figure 2a2, b12) and tetrapolar spindles (Figure 1b), with a characteristic Y- or cruciform X-shaped organization respectively, in some cases confirmed by
-tubulin labelling of spindle poles (Figure 4a25), at cleavage and early blastocyst stages. Also, in a day 3 embryo,
-tubulin labelling revealed three distinct foci at one pole and one at the other pole of a bipolar spindle, suggesting the presence of four centrosomes in an unbalanced arrangement (Figure 4b13). The localization of
-tubulin at the spindle poles of mitotically dividing blastomeres and acetylated tubulin at midbodies during telophase and the spindle poles during both metaphase and anaphase are in agreement with previous reports on mouse and human oocytes (Schatten et al., 1988
; George et al., 1996
).
In humans, the centrosome of the zygote (the organizing centre of the mitotic spindle which is composed of two centrioles) is paternally inherited from the fertilizing sperm (Sathananthan et al., 1991). Following IVF and abnormal dispermic fertilization, three pronuclei are formed and the presence of two centrosomes most frequently results in a tripolar spindle, or less often in the formation of a bipolar spindle, when the supernumerary centrosome remains dormant (Plachot et al., 1989
; Staessen and Van Steirteghem, 1997
; Sathananthan et al., 1999
). Segregation of the three sets of chromosomes on a bipolar spindle results in a uniformly triploid 2-cell embryo. However, tripolar spindle formation results in a mosaic embryo with three equally sized blastomeres at the first mitotic division, due to the random segregation of the sister chromatids of the three haploid sets of chromosomes to the three poles (Palermo et al., 1994
; Staessen and Van Steirteghem, 1997
; Sathananthan et al., 1999
). By analogy, tripolar (or tetrapolar) spindles at cleavage and early blastocyst stages could also result in chromosomal malsegregation and explain the chromosomal chaos identified by multicolour FISH analysis in some cleavage stage embryos (Delhanty et al., 1997
).
With human cells in culture, binucleate cells formed by failure of cytokinesis, which inherit both centrosomes from the previous division, either form a single bipolar spindle at metaphase resulting in two tetraploid daughter cells following mitosis or, if both centrosomes divide synchronously or asynchronously, form abnormal tetrapolar and tripolar spindles which can result in malsegregation of chromosomes to two or more daughter cells (Cimini et al., 1999). As binucleate blastomeres are common at preimplantation stages, we propose that this could be a major pathway resulting in postzygotic chromosomal abnormalities in the human embryo in vitro. We hypothesize therefore that failed or asymmetric cytokinesis results in the formation of binucleate blastomeres which secondarily leads to tetraploidy or spindle pole abnormalities and chromosomal chaos. Subsequent abnormal mitoses in these cells could then lead to higher order polyploidy and, combined with non-disjunction and chromosome loss of single or more chromosomes, this would provide an explanation for all of the different aneuploidies observed at cleavage and blastocyst stages. This model links binucleate cells with the generation of tetraploid and higher order polyploidy and chromosomal chaos and predicts that nuclear and chromosomal abnormalities are interrelated through abnormalities in cytokinesis and spindle formation.
The relatively high incidence of postzygotic chromosomal abnormalities detected by multicolour FISH analysis of cleavage stage embryos led to the hypothesis that, as with some invertebrate and lower vertebrate embryos, cell cycle checkpoints may not operate during cleavage before global activation of the embryonic genome, resulting in genetic instability similar to that observed with human tumour cells (Delhanty and Handyside, 1995). In human embryos, global activation of the embryonic genome occurs on day 3 at the 48-cell stage (Braude et al., 1988
; Taylor et al., 1997
). However, some maternal proteins inherited in the unfertilized oocyte appear to persist throughout preimplantation development (Leese et al., 1991
; Taylor et al., 2001
). Abnormal mitoses in the first three cleavage divisions would therefore exit mitosis and generate daughter cells with abnormal chromosomal complements whereas at the blastocyst stage, a functional spindle assembly checkpoint would minimize their deleterious effects by arresting mitosis until the defect is corrected or by eliminating mitotically arrested cells by apoptosis, essentially as has been suggested for somatic cells (Rieder and Palazzo, 1992
; Musacchio and Hardwick, 2002
). In vertebrate somatic cells and sea-urchin zygotes, mitosis is prolonged 23-fold when monopolar spindles assemble (Sluder and Begg, 1983
; Wang et al., 1983
). However, Sluder et al. (1997)
, who analysed the duration of mitosis in sea-urchin zygotes containing tripolar or tetrapolar spindles, reported that unlike monopolar spindles, the presence of supernumerary spindle poles did not delay anaphase, suggesting that the checkpoint control for metaphaseanaphase transition does not monitor excess spindle poles or bipolar spindle symmetry. The authors concluded that animal cells do not seem to have a checkpoint for the metaphaseanaphase transition independent of the checkpoint that monitors kinetochore attachment to the spindle, and they proposed that the spindle assembly checkpoint is in effect the kinetochore attachment checkpoint.
As with the analogous genetic instability in human tumours, it is difficult to distinguish whether postzgotic nuclear and chromosomal abnormalities are the cause or consequence of developmental arrest. Furthermore, the identification of spindle abnormalities per se does not provide any information about the operation of cell cycle checkpoints, although most nuclei in arrested embryos were in interphase. However, the effects of these abnormalities on the development of human preimplantation embryos in vitro, whether primary or secondary, will depend on the stage at which they occur, the proportion of cells affected and the potential for further division. Clearly, if an abnormality arises in early cleavage then a large proportion of the embryo will be affected, whereas an isolated occurrence at the blastocyst stage may only have a marginal impact and chromosomally abnormal cells may be eliminated by apoptosis. For example, both blastomeres in the arrested 2-cell embryo shown in Figure 3a are tetranucleate. It is possible that failure of cytokinesis occurred in both blastomeres at the equivalent of both the second and third cleavage divisions and that tetrapolar spindles were formed as a result, producing the tetranucleate blastomeres. In this case, therefore, it seems highly unlikely that either blastomere would be viable but also that the nuclear abnormalities are secondary to the primary cause of cytokinetic failure.
Finally, the multipolar spindles observed at the blastocyst stage were in the mural trophectoderm (Figures 2a,b). In the mouse, tetraploid cells mainly contribute to the trophectoderm at the blastocyst stage and are eliminated selectively from the inner cell mass lineage from which the fetus is derived (James et al., 1995). The latter has been proposed as a mechanism for confined placental mosaicism of aneuploid cells in human development (James and West, 1994
). It is possible therefore that binucleate and/or tetraploid blastomeres normally contribute only to extraembryonic lineages and similarly a minority of aneuploid cells may simply be eliminated from the inner cell mass lineage. However, Evsikov and Verlinsky (1998)
and Derhaag et al. (2003)
reported the presence of aneuploid and tetraploid cells in both lineages in human blastocysts. Further research is therefore necessary to determine if this correlates with the presence of spindle abnormalities.
Multipolar spindles and chromosome loss inevitably lead to chromosomal malsegregation and may account for much of the observed postzygotic chromosomal mosaicism in human preimplantation embryos in vitro. Whether spindle abnormalities are limited to in vitro-produced embryos and are linked to parental infertility or culturing conditions, or whether they constitute part of normal development, warrants further investigation. Early identification of abnormal spindles may, however, allow better embryo selection in current IVF programmes. Polarizing microscopy has been employed for non-invasive visualization of the meiotic spindle in unfertilized oocytes to avoid damaging it during ICSI (Wang and Keefe, 2002a,b
). It may therefore be possible to use a similar approach for non-invasive assessment of mitotic spindles in cleavage stage embryos. In combination with scoring for pronuclear morphology at the 1-cell stage (Gianaroli et al., 2003
), this could provide an effective and non-invasive method for selecting viable embryos for transfer by identifying and rejecting those with extensive postzygotic chromosomal abnormalities.
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Acknowledgements |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cimini D, Tanzarella C and Degrassi F (1999) Differences in malsegregation rates obtained by scoring ana-telophases or binucleate cells. Mutagenesis 14, 563568.
Delhanty JDA and Handyside AH (1994) The origin of genetic defects in the human and their detection in the preimplantation embryo. In Charlton HC (ed.) Oxford Reviews of Reproductive Biology, Vol 17. Oxford University Press, Oxford.
Delhanty JDA and Handyside AH (1995) The origin of genetic defects in the human and their detection in the preimplantation embryo. Hum Reprod Update 1, 201215.[CrossRef][ISI][Medline]
Delhanty JD, Harper JC, Ao A, Handyside AH and Winston RM (1997) Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet 99, 755760.[CrossRef][ISI][Medline]
Derhaag JG, Coonen E, Bras M, Bergers Janssen JM, Ignoul-Vanvuchelen R, Geraedts JPM, Evers JLH and Dumoulin JCM (2003) Chromosomally abnormal cells are not selected for the extra-embryonic compartment of the human preimplantation embryo at the blastocyst stage. Hum Reprod 18, 25652574.
Evsikov S and Verlinsky Y (1998) Mosaicism in the inner cell mass of human blastocysts. Hum Reprod 13, 31513155.[Abstract]
Gardner DK and Lane M (2003) Towards a single embryo transfer. Reprod Biomed Online 6, 470481.[Medline]
Gardner DK, Vella P, Lane M, Wagley L, Schlenker T and Schoolcraft WB (1998a) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 69, 8488.[CrossRef][ISI][Medline]
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J and Hesla J (1998b) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 13, 34343440.[Abstract]
George MA, Pickering SJ, Braude PR and Johnson MH (1996) The distribution of alpha- and gamma-tubulin in fresh and aged human and mouse oocytes exposed to cryoprotectant. Mol Hum Reprod 2, 445456.[Abstract]
Gianaroli L, Magli MC, Ferraretti AP, Tabanelli C, Trombetta C and Boudjema E (2002) The role of preimplantation diagnosis for aneuploidies. Reprod Biomed Online 4(Suppl 3), 3136.[Medline]
Gianaroli L, Magli MC, Ferraretti AP, Fortini D and Grieco N (2003) Pronuclear morphology and chromosomal abnormalities as scoring criteria for embryo selection. Fertil Steril 80, 341349.[CrossRef][ISI][Medline]
Griffin DK, Wilton LJ, Handyside AH, Atkinson GH, Winston RM and Delhanty JD (1993) Diagnosis of sex in preimplantation embryos by fluorescent in situ hybridisation. Br Med J 306, 1382.[ISI][Medline]
Hardy K (1999) Apoptosis in the human embryo. Rev Reprod 4, 125134.
Hardy K, Winston RM and Handyside AH (1993a) Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. J Reprod Fertil 98, 549558.[ISI][Medline]
Hardy K, Winston RML and Handyside AH (1993b) Binucleate cells in human preimplantation embryos in vitro: failure of cytokinesis during cleavage. J Reprod Fertil 98, 549558.[ISI][Medline]
Harper JC, Coonen E, Handyside AH, Winston RM, Hopman AH and Delhanty JD (1995) Mosaicism of autosomes and sex chromosomes in morphologically normal, monospermic preimplantation human embryos. Prenat Diagn 15, 4149.[ISI][Medline]
Harrison RH, Kuo HC, Scriven PN, Handyside AH and Ogilvie CM (2000) Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH. Zygote 8, 217224.[CrossRef][ISI][Medline]
Jackson KV, Ginsburg ES, Hornstein MD, Rein MS and Clarke RN (1998) Multinucleation in normally fertilized embryos is associated with an accelerated ovulation induction response and lower implantation and pregnancy rates in in vitro fertilization-embryo transfer cycles. Fertil Steril 70, 6066.[CrossRef][ISI][Medline]
James RM and West JD (1994) A chimaeric animal model for confined placental mosaicism. Hum Genet 93, 603604.[ISI][Medline]
James RM, Klerkx AH, Keighren M, Flockhart JH and West JD (1995) Restricted distribution of tetraploid cells in mouse tetraploiddiploid chimaeras. Dev Biol 167, 213226.[CrossRef][ISI][Medline]
Kligman I, Benadiva C, Alikani M and Munné S (1996) The presence of multinucleated blastomeres in human embryos is correlated with chromosomal abnormalities. Hum Reprod 11, 14921498.
Lamb NE, Feingold E, Savage A, Avramopoulos D, Freeman S, Gu Y, Hallberg A, Hersey J, Karadima G, Pettay D et al. (1997) Characterization of susceptible chiasma configurations that increase the risk for maternal nondisjunction of chromosome 21. Hum Mol Genet 6, 13911399.
Leese HJ, Humpherson PG, Hardy K, Hooper MA, Winston RM and Handyside AH (1991) Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography. J Reprod Fertil 91, 197202.[ISI][Medline]
Lewis CM, Pinel T, Whittaker JC and Handyside AH (2001) Controlling misdiagnosis errors in preimplantation genetic diagnosis: a comprehensive model encompassing extrinsic and intrinsic sources of error. Hum Reprod 16, 4350.
Munné S, Alikani M, Tomkin G, Grifo J and Cohen J (1995a) Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 64, 382391.[ISI][Medline]
Munné S, Sultan KM, Weier HU, Grifo JA, Cohen J and Rosenwaks Z (1995b) Assessment of numeric abnormalities of X, Y, 18, and 16 chromosomes in preimplantation human embryos before transfer. Am J Obstet Gynecol 172, 11911199.[CrossRef][ISI][Medline]
Munné S, Cohen J and Sable D (2002) Preimplantation genetic diagnosis for advanced maternal age and other indications. Fertil Steril 78, 234236.[ISI][Medline]
Munné S, Sandalinas M, Escudero T, Veilla E, Walmsley R, Sadowy S, Cohen J and Sable D (2003) Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod Biomed Online 7, 9197.[Medline]
Musacchio A and Hardwick KG (2002) The spindle checkpoint: structural insights into dynamic signalling. Nature Rev Mol Cell Biol 3, 731741.[CrossRef][ISI][Medline]
Palermo G, Munné S and Cohen J (1994) The human zygote inherits its mitotic potential from the male gamete. Hum Reprod 9, 12201225.[Abstract]
Petzoldt U (1990) Survival of maternal mRNA in anucleate and unfertilized mouse eggs. Eur J Cell Biol 52, 123128.[ISI][Medline]
Plachot M, Mandelbaum J, Junca AM, de Grouchy J, Salat-Baroux J and Cohen J (1989) Cytogenetic analysis and developmental capacity of normal and abnormal embryos after IVF. Hum Reprod 4 (Suppl 8), 99103.[Abstract]
Rieder CL and Palazzo RE (1992) Colcemid and the mitotic cycle. J Cell Sci 102(Pt 3), 387392.[ISI][Medline]
Sathananthan AH, Kola I, Osborne J, Trounson A, Ng SC, Bongso A and Ratnam SS (1991) Centrioles in the beginning of human development. Proc Natl Acad Sci USA 88, 48064810.
Sathananthan AH, Tarin JJ, Gianaroli L, Ng SC, Dharmawardena V, Magli MC, Fernando R and Trounson AO (1999) Development of the human dispermic embryo. Hum Reprod Update 5, 553560.
Schatten G, Simerly C, Asai DJ, Szoke E, Cooke P and Schatten H (1988) Acetylated alpha-tubulin in microtubules during mouse fertilization and early development. Dev Biol 130, 7486.[CrossRef][ISI][Medline]
Sluder G and Begg DA (1983) Control mechanisms of the cell cycle: role of the spatial arrangement of spindle components in the timing of mitotic events. J Cell Biol 97, 877886.[Abstract]
Sluder G, Thompson EA, Miller FJ, Hayes J and Rieder CL (1997) The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry. J Cell Sci 110(Pt 4), 421429.
Staessen C and Van Steirteghem AC (1997) The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod 12, 321327.[Abstract]
Taylor DM, Ray PF, Ao A, Winston RM and Handyside AH (1997) Paternal transcripts for glucose-6-phosphate dehydrogenase and adenosine deaminase are first detectable in the human preimplantation embryo at the three- to four-cell stage. Mol Reprod Dev 48, 442448.[CrossRef][ISI][Medline]
Taylor DM, Handyside AH, Ray PF, Dibb NJ, Winston RM and Ao A (2001) Quantitative measurement of transcript levels throughout human preimplantation development: analysis of hypoxanthine phosphoribosyl transferase. Mol Hum Reprod 7, 147154.
Tesarik J, Cruz-Navarro N, Moreno E, Canete MT and Mendoza C (2000) Birth of healthy twins after fertilization with in vitro cultured spermatids from a patient with massive in vivo apoptosis of postmeiotic germ cells. Fertil Steril 74, 10441046.[CrossRef][ISI][Medline]
Verlinsky Y, Cieslak J and Kuliev A (2002) Preimplantation FISH diagnosis of aneuploidies. Methods Mol Biol 204, 259273.[Medline]
Wang RJ, Wissinger W, King EJ and Wang G (1983) Studies on cell division in mammalian cells VII A temperature-sensitive cell line abnormal in centriole separation and chromosome movement. J Cell Biol 96, 301306.[Abstract]
Wang WH and Keefe DL (2002a) Prediction of chromosome misalignment among in vitro matured human oocytes by spindle imaging with the PolScope. Fertil Steril 78, 10771081.[CrossRef][ISI][Medline]
Wang WH and Keefe DL (2002b) Spindle observation in living mammalian oocytes with the polarization microscope and its practical use. Clon Stem Cells 4, 269276.[CrossRef]
Wang WH, Abeydeera LR, Han YM, Prather RS and Day BN (1999) Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo. Biol Reprod 60, 10201028.
Wang WH, Abeydeera LR, Prather RS and Day BN (2000a) Actin filament distribution in blocked and developing pig embryos. Zygote 8, 353358.[CrossRef][ISI][Medline]
Wang WH, Abeydeera LR, Prather RS and Day BN (2000b) Polymerization of nonfilamentous actin into microfilaments is an important process for porcine oocyte maturation and early embryo development. Biol Reprod 62, 11771183.
Winston NJ, Braude PR, Pickering SJ, George MA, Cant A, Currie J and Johnson MH (1991) The incidence of abnormal morphology and nucleocytoplasmic ratios in 2-, 3- and 5-day human pre-embryos. Hum Reprod 6, 1724.[Abstract]
Submitted on October 19, 2004; accepted on November 3, 2004.