Hum. Reprod. Advance Access originally published online on July 29, 2008
Human Reproduction 2008 23(11):2596-2608; doi:10.1093/humrep/den287
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Comprehensive molecular cytogenetic analysis of the human blastocyst stage
1 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford OX3 9DU, UK 2 Reprogenetics LLC, Livingston, NJ 07039, USA 3 La Jolla IVF, La Jolla, CA 92037, USA 4 Colorado Center for Reproductive Medicine, Lone Tree, CO 80124, USA
5 Correspondence address. E-mail: elpida.fragouli{at}obs-gyn.ox.ac.uk/efragouli{at}hotmail.com
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
BACKGROUND: The high frequency of chromosomal abnormalities observed in human gametes and embryos is unlike that seen in other mammalian species and is of great clinical significance, leading to high rates of pregnancy loss, and live-born children with aneuploid syndromes. Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses during pregnancy, the chromosomal status of blastocysts has been relatively little investigated.
METHODS: A total of 158 good quality blastocysts were examined using micromanipulation, whole genome amplification and comparative genomic hybridization.
RESULTS: From the obtained data, it was evident that the aneuploidy rate (38.8%) is significantly lower for blastocysts than for embryos at earlier stages (51%). However, in many cases, chromosome errors, including monosomy, imbalance affecting the larger chromosomes and complex aneuploidy persisted to this final stage of preimplantation development.
CONCLUSIONS: This study represents the first attempt to gain a detailed insight into the extent and type of chromosome errors seen at the blastocyst stage, using a comprehensive molecular cytogenetic method. Our data suggest that the blastocyst stage does not represent an absolute selective barrier, and that the majority of aneuploid embryos are lost at the time of implantation or shortly thereafter.
Key words: blastocyst/comparative genomic hybridization/chromosome/aneuploidy/preimplantation genetic screening
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Chromosomal abnormalities in human oocytes and preimplantation embryos are extremely common and increase significantly with advancing maternal age. Even women in their 20s and early 30s experience oocyte aneuploidy rates of
20%, while more than half of all oocytes from women over 40 are abnormal (Pellestor et al., 2003
; Fragouli et al., 2006a,b). The frequency of chromosomal abnormality in human oocytes is an order of magnitude greater than seen in other mammalian species and is of great clinical significance, leading to high rates of children born with aneuploid syndromes (e.g. Down syndrome) and also miscarriage. The fact that >70% of first trimester miscarriages are aneuploid emphasizes both the high incidence and lethality of this problem in humans (Menasha et al., 2005).
As well as causing problems for natural conceptions, the prevalence of lethal chromosome abnormalities in human oocytes and embryos provides a likely explanation for the relatively low success rate of assisted reproductive treatment (ART) cycles in our species. The probability of an embryo produced using in vitro fertilization (IVF) implanting in the uterus declines with advancing maternal age, from 21.8% for women less than 38 years to 7.6% for women 41–42 years of age (SART, 2004). It is believed that in most cases aneuploid embryos either arrest prior to transfer to the mother, fail to implant after transfer, or spontaneously abort early in gestation (reviewed in Delhanty, 2005).
Most of the information concerning the chromosome complement of human preimplantation embryos comes from examination of the cleavage stage of development, 3 days after fertilization. Data have been obtained during the course of preimplantation genetic screening (PGS) cycles, in which single cells biopsied from cleavage stage embryos are analyzed using fluorescent in situ hybridization (FISH) (Delhanty et al., 1993; Munné et al., 1993). For clinical PGS, most laboratories use FISH to assess 6–10 chromosomes per cell (Colls et al., 2007; Mantzouratou et al., 2007). Together, the various FISH-based studies have indicated that at least two-thirds of human cleavage stage embryos contain aneuploid cells (Delhanty et al., 1997; Magli et al., 2000; Bielanska et al., 2002; Mantzouratou et al., 2007).
In addition to meiotic chromosome segregation errors, mostly originating from the oocyte, post-zygotic errors are extremely common, especially during the first two or three mitotic divisions (Delhanty et al., 1997; Munné et al., 2002; Katz-Jaffe et al., 2004). Chromosome malsegregation occurring after fertilization results in chromosomal mosaicism, the presence of two or more karyotypically distinct cell lines within the same embryo. While some cleavage stage embryos display low-level mosaicism, others appear to display an intrinsic failure of cellular mechanisms that usually ensure accurate chromosome segregation. Such embryos often have different chromosomal complements in each cell examined and have been termed ‘chaotic mosaics’ due to the apparently random assignment of chromosomes to daughter cells (Delhanty et al., 1997).
Data from multiple FISH studies, carried out in many laboratories around the world, have confirmed the high frequency of aneuploidy and mosaicism in early human embryos. However, it was not possible to confirm the absolute aneuploidy rate, as only a restricted set of chromosomes per cell can be accurately assessed using FISH technology. It was not until the development of comprehensive chromosome screening methods for single cells, based upon the use of whole genome amplification and comparative genomic hybridization (CGH), that the true extent of human embryo aneuploidy was revealed (Wells et al., 1999). Using CGH, 51% of cleavage stage embryos were found to be aneuploid in every cell, while a further 24% contained a mixture of abnormal and normal cells. Only 25% of embryos were composed solely of normal cells (Voullaire et al., 2000; Wells and Delhanty, 2000).
Interestingly, early prenatal tests (conducted between weeks 9 and 14) indicate the presence of an aneuploid fetus in <3% of cases (Snijders et al., 1994). This leaves a window of 60 days during which the vast majority of aneuploid embryos are eliminated. However, the question of precisely when the loss of chromosomally abnormal embryos occurs has not yet been adequately answered. It is unclear whether there is a slow attrition, or whether most aneuploid embryos are lost simultaneously, after failing to negotiate a critical developmental milestone.
Although much is known concerning the aneuploidy rates of oocytes, cleavage stage embryos and fetuses in early pregnancy, the chromosomal status of embryos during the final stage of preimplantation development (the blastocyst stage) has been little investigated. This lack of information is attributed mostly to the fact that routine culture of human IVF embryos to blastocyst stage is a relatively new innovation. It is only in recent years that the use of sequential, stage-specific media combined with ultra-stable low oxygen culture systems has permitted blastocyst culture to be accomplished with high efficiency (Schoolcraft et al., 1999; McArthur et al., 2005).
Reaching and surviving the blastocyst stage presents a number of potential challenges for embryos. By this point, few, if any, of the maternal mRNA transcripts provided by the oocyte persist. Thus, the embryo is reliant upon the expression of its own genome, activated 2 days earlier. For this reason, genetic problems such as aneuploidy are likely to have an increasingly negative affect as preimplantation development continues. Additionally, in order to form a blastocyst, the embryo must successfully undertake the first cellular differentiation [formation of the trophectoderm (TE) and inner cell mass (ICM)]; a subtle process that may be hampered by the inappropriate gene expression that inevitably accompanies aneuploidy.
The few cytogenetic investigations of the blastocyst stage reported to date have mostly analyzed small numbers of blastocysts derived from suboptimal embryos (discarded from IVF treatment cycles) using FISH probes (Evsikov and Verlinsky, 1998; Magli et al., 2000; Sandalinas et al., 2001; Bielanska et al., 2002; Coonen et al., 2004). All these studies suggest that human blastocysts, like cleavage stage embryos, carry significant levels of chromosomal abnormality. However, the frequency of aneuploid embryos and the proportion of abnormal cells in mosaic diploid/aneuploid embryos appears to be reduced compared with the cleavage stage (Evsikov and Verlinsky, 1998; Coonen et al., 2004).
Only one group of researchers has previously attempted to examine the entire chromosome complement of blastocysts (Clouston et al., 1997, 2002). For this purpose, conventional techniques of culture synchronization, disruption of the mitotic spindle and G-banding were employed. This study produced valuable data, but was limited by the very low numbers of complete metaphases obtained from each blastocyst, difficulties in clearly distinguishing individual chromosomes and the high risk of artefactual loss of chromosomes during fixation and spreading. The findings were, however, generally confirmatory of the results obtained during FISH investigations (Clouston et al., 1997, 2002).
We report here results obtained during the comprehensive analysis of 158 good quality blastocysts with the use of CGH. This represents the first attempt to analyze the human blastocyst stage embryo using a comprehensive molecular cytogenetic method.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Details of participating patients
A total of 37 couples with no known chromosome abnormalities consented in donating 158 non-transferred embryos towards this study. These couples were undergoing ART procedures with or without PGS at two different IVF centres: the Colorado Center for Reproductive Medicine, Lone Tree, CO, USA, and the La Jolla IVF, La Jolla, CA, USA. Supplementary Table 1 shows the maternal ages, indications, PGS cycle details and number of embryos donated per couple.
Ethical approval
Donation of embryos from all the patients occurred only after their informed consent. This research work was approved by the Institutional Review Boards of the two IVF Centres.
Ovarian stimulation and culture of embryos to the blastocyst stage
The ovarian stimulation protocol was similar in all Centres, and took place as described in Schoolcraft and Gardner (2000). The culture system for blastocyst growth has been described previously (Gardner et al., 1998). The gas phase for embryo culture was 6% CO2/5% O2. All gamete and embryo manipulations occurred in a pediatric isolate designed to control humidity, temperature and pH fluctuation.
Blastocyst groups
Cytogenetic analysis of 158 embryos cultured to the blastocyst stage took place during the course of this study. All investigated blastocysts were classified to be of good quality, after observation of ICM and TE development, expansion and hatching status took place, as described in Schoolcraft et al. (1999). These blastocysts were divided into three different groups:
Group 1: A total of 12 embryos previously characterized as abnormal after Day 3 biopsy and subsequent PGS FISH analysis were cultured to the blastocyst stage. The PGS was carried out by a commercial PGS provider. The embryos were disaggregated and part of them placed into microcentrifuge tubes for CGH analysis. The remainder of these blastocysts was spread onto microscope slides, and these were examined via FISH. These embryos were donated from five patients (nos 1–5).
Group 2: Similar to the Group 1 blastocysts, the 10 embryos of Group 2 were excluded from transfer after cleavage-stage PGS FISH analysis had identified chromosome abnormalities in biopsied blastomeres. After culture until Day 5, these blastocysts had their ICM and TE separated via micromanipulation and placed into microcentrifuge tubes so that they could be investigated via CGH. Four different patients (nos 6–9) donated the blastocysts of this group.
Group 3: A total of 136 good quality embryos were donated from 28 couples (nos 10–37) undergoing ART procedures without Day 3 PGS. These embryos were cultured until Day 5, and they were either subjected to TE biopsy (119 embryos, 20 patients), or placed intact in microcentrifuge tubes (17 embryos, 8 patients). The embryos in this group were examined solely by CGH.
Disaggregation of blastocysts
Micromanipulation of all blastocysts was necessary so that they could be prepared for CGH and FISH analysis or have their TE separated from the ICM. Initially, all blastocysts were placed in handling medium (MOPS, no protein). Then, the blastocysts of Group 1 were divided in half with the use of a blade (Ultra Sharp Splitting Blades, Bioniche Animal Health, Washington, USA) attached to a micromanipulator. A portion (5–10 cells) of these blastocysts was processed for CGH as described in Fragouli et al. (2006a), and then placed in microcentrifuge tubes, whereas another portion (4–15 cells) was fixed onto microscope slides using a slightly modified Carnoid method (Velilla et al., 2002). The TE and ICM of the Group 2 blastocysts were also separated by mechanical micromanipulation and then placed in microcentrifuge tubes for CGH analysis. Blastocysts belonging to Group 3 were either placed intact in tubes or underwent Day 5 biopsy, and the TE samples obtained this way were then prepared for CGH analysis.
Blastocyst biopsy
Using an inverted Nikon microscope with Hoffman optics (Narishige manipulators and injectors) and laser (Hamilton Thorne), a very small ‘channel’ in the zona pellucida 5 µm wide was created on Day 3 of embryonic development. By Day 5 or 6, a few TE cells from the expanding blastocyst were herniating out of the Day 3 hole. The herniated cells were aspirated into a 30 µm biopsy tool. The laser was fired at the constricted area of cells at the end of the biopsy tool. The group of TE cells was separated from the rest of the blastocyst by pulling them gently.
Fluorescent in situ hybridization
Groups 1 and 2 embryos were subjected to Day-3 PGS using a 10 chromosome FISH screen (13, 14, 15, 16, 17, 18, 21, 22, X and Y). This was undertaken by a commercial laboratory (Shady Grove Center for PGD, USA).
We undertook follow-up Day 5/6 FISH analysis of the Group 1 blastocysts with the use of two sequential rounds, as we described in Munné et al. (1998a). Chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were assessed (standard PGS panel, Reprogenetics LLC, USA).
FISH scoring criteria
Previously published scoring criteria (Munné et al., 1998b) were employed during the analysis of the Group 1 blastocysts. These enabled the differentiation of close signals representing two homologous chromosomes from two domains belonging to a split signal of a single chromosome.
Comparative genomic hybridization
The protocol employed for the comprehensive cytogenetic analysis of the blastocysts was as described and validated previously (Wells et al., 1999; Fragouli et al., 2006b). In brief, initial cell lysis of all blastocyst samples took place by incubating them in 3 µl of proteinase K (PK, 125 µg/ml, Roche, Nutley, USA) and sodium dodecyl sulphate (SDS, 17 mM, Invitrogen, Grand Island, USA) at 37°C for 1 h, followed by an incubation at 95°C for 10 min to inactivate the PK enzyme. Genomic 46,XY DNA was extracted from blood, diluted to a concentration ranging between 0.5 and 1 ng/µl and was used as reference, against which the test blastocyst DNA was to be hybridized and compared. The degenerate oligonucleotide primed-PCR (DOP-PCR) was used for the whole genome amplification of the blastocysts from all groups and also for 46,XY reference DNA amplification. Test and reference DNAs were fluorescently labelled via nick translation, according to the manufacturer’s instructions (Nick translation kit, Abbott, IL, USA). The test blastocyst DNA was labelled in green (Spectrum Green-dUTP, Abbott, IL, USA), whereas the reference 46,XY DNA was labelled in red (Spectrum Red-dUTP, Abbott, IL, USA). Test and reference DNAs co-precipitation, their denaturation, along with that of the slides, and the post-hybridization washes all took place as described previously in Fragouli et al. (2006b). The hybridization time was 72 h.
Microscopy, image analysis and interpretation
Metaphase spreads were observed with the use of an Olympus BX 61 fluorescent microscope with a cooled charge-coupled device (CCD) system, and filters for the fluorochromes used. Ten metaphases were captured on average per hybridization. Analysis and interpretation of the captured images was feasible with the use of the Cytovision CGH software (Version 3.9, Applied Imaging, San Jose, USA) that converted fluorescent intensities into a red–green ratio for each chromosome. Equal sequence copy number between the test and reference DNAs was seen as no fluctuation of the ratio profile from 1:1. Test sample under-representation was seen as fluctuation of the ratio profile in favour of the red colouration (below 0.80), whilst test sample over-representation was seen as fluctuation of the ratio profile towards the green colouration (above 1.20). Such fluctuations were, respectively, scored as losses or gains in the test sample, compared with the reference sample. This study was carried out blindly, as far as the Groups 1 and 2 blastocysts were concerned. Specifically, during CGH analysis of the Group 1 blastocysts, their corresponding FISH results were not known. Additionally, the TE and ICM samples of the Group 2 blastocysts were coded, and CGH results were compared only when this part of the study was completed.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Thirty-seven couples undergoing ART procedures agreed to donate their untransferred embryos for this study (Supplementary Table 1). Nine of the couples also previously had their embryos tested on Day-3 using FISH for the purpose of PGS. A total of 158 embryos reached the blastocyst stage after being cultured until Days 5–6. The embryos were divided into three groups, as described in the Materials and Methods section, and subjected to comprehensive chromosome examination employing CGH. A subset of 12 blastocysts were also tested using FISH analysis of at least nine chromosomes, in order to verify results obtained using CGH and reveal chromosomal mosaicism.
Comparison of cytogenetic results obtained after FISH and CGH analysis of human blastocysts
Twelve embryos from five patients were examined at the blastocyst stage using both FISH and CGH (Table I). The embryos were generated by five couples who underwent IVF or ICSI in combination with PGS (chromosomes 13, 15, 16, 17, 18, 21, 22, X, Y screened using single blastomere FISH). The average maternal age was 34.6 years (range 24–41 years). The embryos investigated during this part of the study had been classified abnormal after PGS and consequently were not eligible for transfer (Group 1 in Materials and Methods). All the embryos examined had reached the blastocyst stage prior to being processed for FISH and CGH on Day 5. No arrested embryos were examined.
|
The mean number of cells assessed by FISH was 8.8 per blastocyst, while the number of cells tested by CGH was estimated to be 5–10 (equivalent to a clinical blastocyst biopsy). FISH and CGH analysis were carried out in a blinded fashion, by different operators in different laboratories. Both analyses were successful for all 12 embryos, allowing direct comparison of blastocyst FISH and CGH results. The chromosomes examined by FISH on Day 5 were identical to those assessed for the purpose of Day-3 PGS (see above) with the exception of chromosome 14, which was not screened on Day 5.
FISH and CGH results were in total agreement for 10 out of the 12 embryos, whereas only the sex was confirmed for the remaining two (1-1, 1-2). Both of these embryos were classified as mosaic diploid/aneuploid. In each of these cases, FISH had yielded suboptimal results, and data were available from very few cells (n = 4 for both). Of the 10 blastocysts for which FISH and CGH agreed, five were classified as being normal (three females, two males), three as uniformly aneuploid (three males) and two as aneuploid mosaic (one male, one female).
Comparison of cytogenetic results obtained after CGH analysis of TE and ICM samples
Ten embryos (defined as Group 2 in Materials and Methods) were donated by four couples undergoing ART procedures in combination with blastomere PGS for various indications (see Supplementary Table 1). The average age of the female partners was 32.5 years (range: 26–39 years). After being diagnosed aneuploid by Day-3 FISH, the embryos were cultured until Days 5–6. Upon reaching the blastocyst stage TE and ICM cells were separated mechanically and a similar amount of material (5–10 cells) was placed in microcentrifuge tubes for CGH analysis.
Agreement of CGH results from TE and corresponding ICM cells was seen for all 10 embryos (Table II). Of these, four embryos were classified as normal (two females, two males) on Days 5–6, while chromosome abnormalities were scored for the remaining 6. The fact that results were identical for samples of TE and ICM suggests that data obtained from TE biopsies can generally be considered diagnostic of ICM aneuploidy.
|
Comprehensive cytogenetic analysis of an unselected group of human blastocysts
During the final part of this study, we employed CGH for the complete cytogenetic investigation of 136 good quality blastocysts (Group 3 embryos), donated from 28 couples. These patients were all undergoing ART procedures without Day-3 PGS (see Supplementary Table 1 for indications). Hence, there was no prior selection according to chromosome constitution for any of the embryos in this group. The average maternal age was 36.5 years, with the female partner’s ages ranging from 29 to 45 years. Seventeen of the Group 3 blastocysts were placed intact in microcentrifuge tubes for CGH analysis, while the remaining 119 underwent TE biopsy, and 3–10 of their cells were examined cytogenetically. A summary of the cytogenetic results obtained during the investigation of the Group 3 blastocysts is shown in Table III. Fig. 1 demonstrates the analyses and the resulting interpretations for the 19-2 and 24-5 blastocysts.
|
|
CGH yielded results for 126 of the 136 Group 3 embryos, and therefore showed an efficiency of 93%. Forty-nine embryos were classified as being aneuploid, leading to an overall abnormality rate of 38.8%. It is possible that the true aneuploidy rate is slightly higher than this figure, due to the inability of CGH to detect ploidy changes (e.g. triploidy, tetraploidy etc.). However, such abnormalities affect <4% of Day-3 embryos and probably even fewer blastocysts, so the difference is expected to be small (S. Munné personal communication). Tetraploid cells increase in frequency as preimplantation development progresses. However, the appearance of such cells is generally considered to be a hallmark of trophoblast differentiation and is probably not diagnostically or clinically relevant (Bielanska et al., 2002
). Aneuploidies involving almost all chromosomal groups (A–F) were seen during the later stages of human preimplantation development. Table IV summarizes the chromosome errors detected at the blastocyst stage during this study. The data for this table were taken only from the Group 3 embryos, as unlike Groups 1 and 2 embryos, these represent an unbiased sample (i.e. no prior selection based upon Day-3 PGS).
|
The sex chromosomes (X/Y) were most frequently involved in errors (13 blastocysts). Eight of the blastocysts with errors involving the sex chromosomes were predicted have one extra sex chromosome, resulting in 47,XXY or 47,XYY karyotypes. However, it is possible that one or more of these embryos may have been triploid (69,XXY or 69,XYY), as CGH cannot accurately distinguish between these two possibilities.
The autosomes most often found to be abnormal were chromosomes 22 and 21 (10 and 8 blastocysts, respectively), chromosomes 15, 18 and 19 (6 blastocysts each), 14, 1, 8, 11, 2, 13, 16, and 20 (2–5 blastocysts each), and 6, 7, 10, and 12 (one blastocyst each). In total, 81 whole chromosome anomalies were observed, of which 35 were trisomies and 46 were monosomies. Additionally, two structural abnormalities, both in the form of gains, were detected for blastocysts 23-2 and 23-5. Interestingly, both these blastocysts were generated from couple no. 23. Gains of the entire q arm of chromosome 1 (1q21.1-q44) were scored in both cases.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
By employing a comprehensive cytogenetic method, we were able to obtain a more complete picture of the extent of chromosome abnormalities during the final phase of human preimplantation development than has previously been possible. Important information was obtained concerning the fate of aneuploid gametes and embryos, which are extremely common in humans. Additionally, the data collected provide essential pre-clinical validation of a novel approach for PGS based upon CGH analysis of cells biopsied from the TE. Such an approach permits accurate screening of all chromosomes and removes one of the most difficult steps in the current PGS procedures—fixation of cells on a microscope slide.
One hundred and thirty-six of the embryos examined were not subject to any prior selection and can be considered a representative sample of human blastocysts (Group 3). Of the 126 blastocysts, which yielded CGH results, 49 (38.8%) embryos were characterized as being aneuploid. This rate is comparable to that found by Clouston et al. (2002) using G-banding to assess blastocyst cells in metaphase, and probably corresponds to the true aneuploid frequency for human embryos during the final stage of preimplantation development (36.5 years mean maternal age). Previous CGH studies conducted at the cleavage stage (Day 3) revealed that 51% of embryos are aneuploid in all cells, 32% due to meiotic error, with a further 19% devoid of normal cells due to mitotic errors occurring after fertilization (Voullaire et al., 2000, 2007; Wells and Delhanty, 2000). The aneuploidy rate we observed at the blastocyst stage (38.8%) was lower, indicating a gradual loss of aneuploid embryos between Days 3 and 5.
The blastocysts assessed in this study were derived from patients undergoing IVF treatment and it remains possible that the aneuploidy rate for fertile patients could be somewhat different. However, division of the patients into various subcategories based upon infertile aetiology revealed similar incidence of aneuploidy in all groups, despite very different reasons for IVF treatment (male factor, various forms of ovarian dysfunction, structural problems etc). These near identical abnormality rates suggest that aneuploidy is not associated with a specific form of infertility in these patients.
Comparison of FISH and CGH analysis performed on the same blastocysts (Group 1) demonstrated complete agreement for embryo sex chromosome constitution in all cases. Autosome copy numbers were also confirmed for all embryos except two. The samples with discordant results both yielded suboptimal FISH results with poor fixation and data available from very few cells (n = 4 in both cases). The small number of cells assessed increases the likelihood that a cytogenetically distinct subpopulation of cells could have been sampled from a mosaic embryo. Additionally, poor fixation is well known to increase the risk of technical problems affecting FISH analysis, such as hybridization failure and misinterpretation due to high background levels and low signal intensities. In some cases, two CGH results were available from the same blastocyst (Group 2), and these were concordant for all embryos tested (10 of 10).
The principal advantage of CGH is its ability to screen the entire chromosome complement, allowing it to reveal abnormalities of chromosomes not routinely screened using FISH. For example, both FISH and CGH indicated that blastocyst 5-1 was monosomic for chromosome 22. However, CGH revealed an additional aneuploidy affecting chromosome 8, a chromosome rarely included in FISH screens.
Several FISH probe combinations have been applied to human cleavage stage embryos, with varying numbers of chromosomes examined (Colls et al., 2007; Mantzouratou et al., 2007). Most probe combinations have targeted the sex chromosomes and the smaller autosomes (chromosomes 13–22), as these are most frequently involved in abnormalities seen in spontaneous abortions, prenatal samples and live-born offspring. Larger chromosomes (i.e. chromosomes 1–12) have been relatively ignored by FISH-based studies.
The current study demonstrates that many aneuploidies affecting larger autosomes can persist to the blastocyst stage and probably survive until the time of implantation or shortly thereafter. Indeed, errors affecting chromosome 2 represented one of the most common cytogenetic anomalies detected during this study. Whether these errors were of meiotic or mitotic origin is unclear. However, in a separate study of over 400 oocytes using CGH, malsegregation of large chromosomes was observed at a similar frequency, suggesting that in most cases these forms of aneuploidy originate during meiosis (Fragouli et al. 2006a,b; Fragouli and Wells, unpublished).
It may be significant that the total blastocyst aneuploidy rate recorded during this study is very close to the total gamete aneuploidy rate (oocyte+sperm) observed for patients with the same average maternal age (i.e. 36.5 years). Such an observation may suggest that the majority of aneuploidies that survive to the blastocyst stage are those of meiotic in origin. This would distinguish blastocysts from cleavage stage embryos, 13–20% of which are aneuploid in every cell due to the presence of multiple mitotic errors (i.e. chaotic mosaicism). It is probable that cell–cell interactions are impaired in highly mosaic embryos, interfering with the coordinated reorganization and differentiation necessary for blastocyst formation, ultimately leading to embryo demise before the blastocyst stage.
As well as the errors involving large chromosomes a variety of monosomies were also identified. Autosomal monosomies are common at the cleavage stage, but are rarely identified in spontaneous abortion material and are all but absent from second trimester prenatal samples (Simoni et al., 1986; Simpson, 1990; Wolstenholme, 1996). There has been some controversy in the literature concerning the point at which monosomic embryos are lost. Sandalinas et al. (2001) followed the developmental progress of embryos tested for aneuploidy on Day 3 using a nine chromosome FISH screen. The only autosomal monosomy they found to survive to the blastocyst stage was monosomy 21. Similarly, Clouston et al. (2002) found no monosomic embryos in a series of blastocysts analyzed using conventional G-banding. These studies suggest loss of most monosomies around the morulla stage (Day 4). However, results from other studies involving FISH analysis of blastocysts have revealed that several autosomal monosomies are capable of forming morphologically normal blastocysts (Magli et al., 2000; Stephenson et al., 2002; Rubio et al., 2003). During the current study, we identified 31 unselected (Group 3) good quality blastocysts carrying monosomies compared with 27 with trisomies. This finding confirms that the morulla stage is not an insurmountable boundary to the further development of monosomic embryos.
Differences between studies in terms of monosomy detection might be explained by differences in the chromosomes screened. Even the most detailed of the previous FISH studies would have failed to detect half of the monosomic chromosomes observed in the current study, potentially leading to an underestimate of monosomic survival. Additionally, no two blastocyst studies have employed precisely the same culture method, raising the possibility that certain culture systems provide better support for highly abnormal embryos, improving their chances of successful development to the blastocyst stage. The blastocyst culture method employed during the current study is believed to provide a closer approximation to the in vivo environment than earlier studies.
In addition to the detection of aneuploidies involving entire chromosomes, CGH is able to detect structural anomalies resulting in loss/gain of chromosomal material. Examples of chromosome breakage were observed in three blastocysts from Groups 1 and 2. The ability of CGH to assess the entire length of each chromosome represents a significant advantage over FISH. The probes used for FISH hybridize to small chromosomal regions, from which the presence of the rest of the chromosome is assumed, sometimes incorrectly.
In one case, CGH detected two structural errors, both involving breakage and duplication of a large segment of chromosome 1 (1q21.1-q44). These partial aneuploidies were seen in two different blastocysts (23-2, 23-5) generated from the same couple (patient 23). None of the patients who donated their blastocysts were known to carry chromosome abnormalities. However, a formal karyotype was not available in all cases. It is therefore possible that patient 23, or her partner, was a balanced carrier of a chromosome rearrangement involving the long arm of chromosome 1, resulting in the generation of abnormal gametes and embryos.
Structural abnormalities were also observed during Clouston’s G-banding investigation of human blastocysts (Clouston et al., 1997). Interestingly in both their study and ours, these partial aneuploidies were seen in embryos characterized as being complex abnormal and possibly chaotic. It seems that highly abnormal embryos tend to have a combination of whole chromosome and segmental anomalies, implying the existence of a common mechanism leading to chromosome breakage and the generation of highly mosaic or chaotic embryos. One such mechanism could be the lagging of chromosomes on the spindle generating breakage during cytokinesis. This may be associated with breakage-bridge fusion cycles in mitotic division, while they appear suppressed in female meiosis (Koehler et al., 2002).
Several cellular pathways protect against a combination of both malsegregation and breakage of chromosomes, e.g. the G2-M cell cycle checkpoint. It is possible that the rapid cell divisions occurring at the blastocyst stage necessitate a relaxation of such checkpoints, increasing the risk of chromosome segregation/breakage errors occurring. Alternatively rapid divisions could lead to a depletion of critical nutrients required for chromosomal integrity. For example, deficiency of folate or nucleoside precursors leads to replication stalling and eventually chromosome fragmentation and aneuploidy. The fact that many of the breakpoints observed in embryos with chromosome fragmentation correspond to known fragile sites (regions of the genome prone to stalling of replication forks) argues in favour of such a mechanism.
The average age of the female patients contributing embryos to Group 3 was calculated to be 36.5 years. Dividing this group of women into two sub-groups, an older (37 years or more, mean age: 39.35 years) and a younger (36 years or less, mean age: 32 years), revealed a significantly higher blastocyst aneuploidy rate for the older group (48.31% versus 16.2%, P = 0.0015, 
2 with Yates correction). It is also noteworthy that the six embryos found to have complex aneuploidy (i.e. imbalance affecting three or more entire chromosomes) were all derived from patients in the older group. It has been shown that the frequency of multiple chromosomal aberrations increases more dramatically with advancing maternal age compared to single trisomies (reviewed in Hassold et al., 2007). These results indicate that even though embryos generated from older women may reach the blastocyst stage, they have an elevated risk of carrying one or more chromosome errors when compared with embryos from younger women.
The presence of complex chromosome abnormalities at the blastocyst stage was somewhat surprising, given the severity of the anomalies recorded. The most extreme example was that of blastocyst no. 24-5 which contained eleven aneuploid chromosomes. It is very likely that embryos with such extensive aneuploidy are mosaic, their development supported by populations of less severely impaired cells. A small number of ‘chaotic’ blastocysts have been observed previously (Sandalinas et al., 2001; Coonen et al., 2004; Bielanska et al., 2005), but such embryos tend to be developmentally compromised being composed of significantly fewer cells than typical blastocysts (Sandalinas et al., 2001).
Out of a total of 22 embryos classified as abnormal after Day-3 PGS using FISH, nine (41%) (five from Group 1 and four from Group 2) were characterized as being diploid normal on Days 5–6 using CGH. Similar findings were described in the study of Li et al., (2005), during which 281 embryos identified as aneuploid on Day 3 were cultured until Day 6 and then reanalyzed. In that study, twenty-two (40%) of the 55 embryos that formed blastocysts were characterized as diploid after FISH analysis of chromosomes 13, 18, 21, X and Y (Li et al., 2005).
While these results appear to indicate a high error rate for PGS, it is important to note that a significant ascertainment bias is in operation. The embryos analyzed have been cultured for an additional 2–3 days following PGS, with only those reaching the blastocyst stage subjected to CGH analysis. Abnormal embryos have a greater probability of undergoing developmental arrest, thus extended culture increases the relative proportion of normal embryos (Staessen et al., 2004). Previous studies have indicated that the error rate of PGS can be as low as 3%, provided that appropriate methods are used (Colls et al., 2007). Unfortunately, no details concerning the PGS method were available to us on this occasion, so technical issues could not be assessed. Although difficult to quantify precisely, it seems to be the case that a minority of embryos diagnosed aneuploid on Day 3 are capable of forming euploid blastocysts.
The most likely explanation for the differences in the cytogenetic constitution of embryos between Days 3 and 5/6 is preferential growth of euploid cells in embryos containing a mixture of normal and abnormal cells. Previous CGH studies have suggested that 24% of human cleavage stage embryos are euploid/aneuploid mosaics (Voullaire et al., 2000; Wells and Delhanty, 2000). It is likely that conventional PGS methods accurately classify embryos that are either entirely euploid or aneuploid in every cell (including aneuploid mosaics). However, the diagnosis and developmental fate of cleavage stage embryos composed both of normal and aneuploid cells remains uncertain.
Several studies have shown that mosaic embryos can form good quality blastocysts (Sandalinas et al., 2001; Coonen et al., 2004 Bielanska et al., 2005). Additionally, Evsikov and Verlinsky (1998) observed a decrease in the proportion of abnormal cells in mosaic embryos by Day 6 compared with earlier stages of development. This suggests that in some mosaic embryos euploid cells have a growth advantage over aneuploid cells, and may end up forming the majority of the embryo and perhaps ultimately creating a chromosomally normal fetus (Wells and Delhanty, 2000; Coonen et al., 2004). Although not conclusive, our data are also consistent with survival to the blastocyst stage and progressive normalization of a proportion of diploid/aneuploid mosaic embryos.
There also exists the possibility that abnormal embryos undergo self-correction, perhaps by extruding micronuclei with extra chromosomes, or by duplication of chromosomes which were monosomic. Self-correction of monosomic and trisomic conceptions has been previously recorded, sometimes resulting in uniparental disomy or confined placental mosaicism (Wolstenholme, 1996; Robinson et al., 1997). However, such events are generally considered to be rare.
The high rate of aneuploidy detected at the blastocyst stage in this study indicates that screening Day-5 in vitro fertilized embryos for chromosome imbalance prior to transfer to the uterus could be a useful approach for identifying high viability embryos. The transfer of blastocysts in the context of IVF treatment is associated with higher implantation rates compared with transfer of embryos at earlier developmental stages. However, it is clear that even at this stage, lethal aneuploidies are relatively common. We speculate that the preferential transfer of chromosomally normal blastocysts could significantly boost implantation and pregnancy rates per embryo transfer.
We have demonstrated concordance between the results obtained after CGH analysis of TE and ICM samples derived from the same blastocyst. This indicates that it should be possible to accurately assess the chromosome constitution of blastocysts by examining cells biopsied from the TE. This finding agrees with the observations of Evsikov and Verlinsky (1998), who found that aneuploid and normal cells were evenly distributed in the TE and ICM in human blastocysts.
Chromosomes belonging to almost all groups were scored as abnormal during the analysis of the Group 3 blastocysts, with the sex chromosomes (X/Y), 21, 22, 19, 20 and 2 being among the most frequently affected. Importantly, chromosomes 19, 20 and 2 are not usually examined by current PGS protocols. If the Group 3 blastocysts were to be examined with the use of FISH for nine chromosomes (13, 15, 16, 17, 18, 21, 22, X, Y), as is typically employed for PGS, 30/83 (36%) of the numerical abnormalities would not have been detected and 9/49 (18.4%) abnormal embryos would have been wrongly categorized ‘normal’.
Unlike conventional Day-3 PGS, analysis at the blastocyst stage allows 5–10 cells to be biopsied without harm to the embryo (de Boer et al., 2004; Kokkali et al., 2007). Not only does the increased number of cells available provide a robust test (94% of samples yielded results during this study), but diagnostic issues of mosaicism are also reduced. CGH provides an ‘average view’ of the biopsied cells and in most cases aneuploidy is not detected unless it is present in more than one-third of the cells.
It could be argued that the failure to detect low-level mosaicism is a disadvantage of PGS at the blastocyst stage using CGH. However, we hypothesize that this type of mosaicism is not of clinical relevance. Abnormal cells in diploid/aneuploid mosaic embryos decline in frequency from Days 3 to 6 and it is likely that this trend continues after implantation, resulting in a normal fetus in most cases.
An important drawback of PGS using CGH is the length of the protocol, necessitating the cryopreservation of biopsied blastocysts and transfer in a subsequent cycle. However, recent studies involving Day 5 biopsy, cryopreservation and genetic analysis have given encouraging results as far as viability and implantation potential of biopsied blastocysts after thawing are concerned (de Boer et al., 2004; McArthur et al., 2005; Kokkali et al., 2007). Furthermore, the freezing of biopsied blastocysts and their transfer in a subsequent cycle could potentially enable a better synchronization between the embryo and the uterus, enhancing implantation and pregnancy.
Our suggested approach of chromosome screening of blastocysts via CGH is obviously not appropriate for patients with poor in vitro embryo development. It could, however, be a powerful selection tool for IVF patients that produce multiple (>3) blastocysts of good morphology, facilitating single embryo transfer. By transferring just one euploid blastocyst per ART cycle, it will be possible to dramatically reduce the incidence of twin and triplet pregnancies, which are associated with greatly elevated risks of complications for both the mother and children. Methods for the identification of viable embryos will be key to maintaining high pregnancy rates while reducing the number of embryos transferred.
|
Supplementary Data |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Supplementary data are available at http://humrep.oxfordjournals.org/.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Institutional funding; Reprogenetics LLC; Colorado Center for Reproductive Medicine.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
The authors would like to thank all the patients who donated their embryos to this study, and the staff of the collaborating Centers, in particular Jessica Filipovits and John Stevens, for their valuable help. We would also like to help the ‘Once Upon a Time Foundation’ for their generous support.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary DataFundingAcknowledgementsReferences |
|---|
Bielanska M, Tan SL, Ao A. High rate of mixoploidy among human blastocysts cultured in vitro. Fertil Steril (2002) 78:1248–1253.[CrossRef][Web of Science][Medline]
Bielanska M, Jin S, Bernier M, Tan SL, Ao A. Diploid-aneuploid mosaicism in human embryos cultured to the blastocyst stage. Fertil Steril (2005) 84:336–342.[CrossRef][Web of Science][Medline]
Clouston HJ, Fenwick J, Webb AL, Herbert M, Murdoch A, Wolstenholme J. Detection of mosaic and non-mosaic chromosome abnormalities in 6- to 8-day old human blastocysts. Hum Genet (1997) 101:30–36.[CrossRef][Web of Science][Medline]
Clouston HJ, Herbert M, Fenwick J, Murdoch AP, Wolstenholme J. Cytogenetic analysis of human blastocysts. Prenat Diagn (2002) 22:1143–1152.[CrossRef][Web of Science][Medline]
Colls P, Escudero T, Cekleniak N, Sadowy S, Cohen J, Munné S. Increased efficiency of preimplantation genetic diagnosis for infertility using "no result rescue". Fertil Steril (2007) 88:53–61.[CrossRef][Web of Science][Medline]
Coonen E, Derhaag JG, Dumoulin JC, van Wissen LC, Bras M, Janssen M, Evers JL, Geraedts JP. Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos. Hum Reprod (2004) 19:316–324.
de Boer KA, Catt JW, Jansen RP, Leigh D, McArthur S. Moving to blastocyst biopsy for preimplantation genetic diagnosis and single embryo transfer at Sydney IVF. Fertil Steril (2004) 82:295–298.[CrossRef][Web of Science][Medline]
Delhanty JD. Mechanisms of aneuploidy induction in human oogenesis and early embryogenesis. Cytogenet Genome Res (2005) 111:237–244.[CrossRef][Web of Science][Medline]
Delhanty JD, Griffin DK, Handyside AH, Harper J, Atkinson GH, Pieters MH, Winston RM. Detection of aneuploidy and chromosomal mosaicism in human embryos during preimplantation sex determination by fluorescent in situ hybridisation, (FISH). Hum Mol Genet (1993) 2:1183–1185.
Delhanty JD, Harper JC, Ao A, Handyside AH, Winston RM. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Hum Genet (1997) 99:755–760.[CrossRef][Web of Science][Medline]
Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod (1998) 13:3151–3155.
Fragouli E, Wells D, Doshi A, Gotts S, Harper JC, Delhanty JD. Complete cytogenetic investigation of oocytes from a young cancer patient with the use of comparative genomic hybridisation reveals meiotic errors. Prenat Diagn (2006) a 26:71–76.[CrossRef][Web of Science][Medline]
Fragouli E, Wells D, Thornhill A, Serhal P, Faed MJ, Harper JC, Delhanty JD. Comparative genomic hybridization analysis of human oocytes and polar bodies. Hum Reprod (2006) b 21:2319–2328.
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod (1998) 13:3434–3340.
Hassold T, Hall H, Hunt P. The origin of human aneuploidy: where we have been, where we are going. Hum Mol Genet (2007) 16:R203–R208.
Katz-Jaffe MG, Trounson AO, Cram DS. Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability. Mol Hum Reprod (2004) 10:143–147.
Koehler KE, Millie EA, Cherry JP, Burgoyne PS, Evans EP, Hunt PA, Hassold TJ. Sex-specific differences in meiotic chromosome segregation revealed by dicentric bridge resolution in mice. Genetics (2002) 162:1367–1379.
Kokkali G, Traeger-Synodinos J, Vrettou C, Stavrou D, Jones GM, Cram DS, Makrakis E, Trounson AO, Kanavakis E, Pantos K. Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study. Hum Reprod (2007) 22:1443–1449.
Li M, DeUgarte CM, Surrey M, Danzer H, DeCherney A, Hill DL. Fluorescence in situ hybridization reanalysis of day-6 human blastocysts diagnosed with aneuploidy on day 3. Fertil Steril (2005) 84:1395–1400.[CrossRef][Web of Science][Medline]
Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod (2000) 15:1781–1786.
Mantzouratou A, Mania A, Fragouli E, Xanthopoulou L, Tashkandi S, Fordham K, Ranieri DM, Doshi A, Nuttall S, Harper JC, et al. Variable aneuploidy mechanisms in embryos from couples with poor reproductive histories undergoing preimplantation genetic screening. Hum Reprod (2007) 22:1844–1853.
McArthur SJ, Leigh D, Marshall JT, de Boer KA, Jansen RP. Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts. Fertil Steril (2005) 84:1628–1636.[CrossRef][Web of Science][Medline]
Menasha J, Levy B, Hirschhorn K, Kardon NB. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med (2005) 7:251–263.[Web of Science][Medline]
Munné S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod (1993) 8:2185–2191.
Munné S, Márquez C, Reing A, Garrisi J, Alikani M. Chromosome abnormalities in embryos obtained after conventional in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril (1998) a 69:904–908.[CrossRef][Web of Science][Medline]
Munné S, Márquez C, Magli C, Morton P, Morrison L. Scoring criteria for preimplantation genetic diagnosis of numerical abnormalities for chromosomes X, Y, 13, 16, 18 and 21. Mol Hum Reprod (1998) b 4:863–870.
Munné S, Sandalinas M, Escudero T, Márquez C, Cohen J. Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect. Reprod Biomed Online (2002) 4:223–232.[Medline]
Pellestor F, Andréo B, Arnal F, Humeau C, Demaille J. Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes. Hum Genet (2003) 112:195–203.[CrossRef][Web of Science][Medline]
Robinson WP, Barrett IJ, Bernard L, Telenius A, Bernasconi F, Wilson RD, Best RG, Howard-Peebles PN, Langlois S, Kalousek DK. Meiotic origin of trisomy in confined placental mosaicism is correlated with presence of fetal uniparental disomy, high levels of trisomy in trophoblast, and increased risk of fetal intrauterine growth restriction. Am J Hum Genet (1997) 60:917–927.[Web of Science][Medline]
Rubio C, Simón C, Vidal F, Rodrigo L, Pehlivan T, Remohí J, Pellicer A. Chromosomal abnormalities and embryo development in recurrent miscarriage couples. Hum Reprod (2003) 18:182–188.
Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munné S. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod (2001) 16:1954–1958.
Schoolcraft WB, Gardner DK. Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril (2000) 74:482–486.[CrossRef][Web of Science][Medline]
Schoolcraft WB, Gardner DK, Lane M, Schlenker T, Hamilton F, Meldrum DR. Blastocyst culture and transfer: analysis of results and parameters affecting outcome in two in vitro fertilization programs. Fertil Steril (1999) 72:604–609.[CrossRef][Web of Science][Medline]
Simoni G, Gimelli G, Cuoco C, Romitti L, Terzoli G, Guerneri S, Rossella F, Pescetto L, Pezzolo A, Porta S, et al. First trimester fetal karyotyping: one thousand diagnoses. Hum Genet (1986) 72:203–209.[CrossRef][Web of Science][Medline]
Simpson JL. Incidence and timing of pregnancy losses: relevance to evaluating safety of early prenatal diagnosis. Am J Med Genet (1990) 35:165–173.[CrossRef][Web of Science][Medline]
Snijders RJ, Holzgreve W, Cuckle H, Nicolaides KH. Maternal age-specific risks for trisomies at 9-14 weeks’ gestation. Prenat Diagn (1994) 14:543–552.[Web of Science][Medline]
Society of assisted reproductive technology and the American society for reproductive medicine. Assisted reproductive technology in the United States: 2000 results generated from the American society of reproductive medicine/ Society of assisted reproductive technology registry. Fertil Steril (2004) 81:1207–1220.[CrossRef][Web of Science][Medline]
Staessen C, Platteau P, Van Assche E, Michiels A, Tournaye H, Camus M, Devroey P, Liebaers I, Van Steirteghem A. Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial. Hum Reprod (2004) 19:2849–2858.
Stephenson MD, Awartani KA, Robinson WP. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage: a case-control study. Hum Reprod (2002) 17:446–451.
Velilla E, Escudero T, Munné S. Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online (2002) 4:210–217.[Medline]
Voullaire L, Slater H, Williamson R, Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet (2000) 106:210–217.[CrossRef][Web of Science][Medline]
Voullaire L, Collins V, Callaghan T, McBain J, Williamson R, Wilton L. High incidence of complex chromosome abnormality in cleavage embryos from patients with repeated implantation failure. Fertil Steril (2007) 87:1053–1058.[CrossRef][Web of Science][Medline]
Wells D, Delhanty JD. Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod (2000) 6:1055–1062.
Wells D, Sherlock JK, Handyside AH, Delhanty JD. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation. Nucleic Acids Res (1999) 27:1214–1218.
Wolstenholme J. Confined placental mosaicism for trisomies 2, 3, 7, 8, 9, 16, and 22: their incidence, likely origins, and mechanisms for cell lineage compartmentalization. Prenat Diagn (1996) 16:511–524.[CrossRef][Web of Science][Medline]
Submitted on April 1, 2008; resubmitted on May 20, 2008; accepted on June 30, 2008.
CiteULike 
Connotea 
Del.icio.us What's this?
Related articles in Hum. Reprod.:
- Editor's Choice
- André Van Steirteghem
Hum. Reprod. 2008 23: 2391.[Extract] [FREE Full Text]
This article has been cited by other articles:
|
|
 |
|
  M.G. Katz-Jaffe, S. McReynolds, D.K. Gardner, and W.B. Schoolcraft The role of proteomics in defining the human embryonic secretome Mol. Hum. Reprod., May 1, 2009; 15(5): 271 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wells, S. Alfarawati, and E. Fragouli Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH Mol. Hum. Reprod., December 1, 2008; 14(12): 703 - 710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C.J.M. Fauser Preimplantation genetic screening: the end of an affair? Hum. Reprod., December 1, 2008; 23(12): 2622 - 2625. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||