Low level chromosome instability in embryonic cells of primary aneuploid mice

Karyotypic analyses of Down syndrome patients have identified a low level of chromosome mosaicism, suggesting that the primary aneuploid status of the cells promotes further chromosomal segregation errors. Sycp3-null female mice produce aneuploid oocytes, which after fusion with normal haploid sperm, result in offspring with systemic whole chromosome, aneuploid embryo cells. Using the Sycp3-null female as a model, we observe an increase in the number of embryonic cells at E7.0 that exhibit abnormal chromosomal bridges at the anaphas estage of mitosis. This result suggests that global changes in gene expression patterns resulting from primary aneuploidy can affect mitotic chromosome segregation, resulting in a low level of chromosomal instability. The increased level of chromosomal instability could in the absence of mitotic checkpoints, lead to chromosomal mosaicism within the adult organism, as seen in Down syndrome patients.     

Aneuploidy in the human cleavage stage embryo

The cleavage stage embryo (days 1-3) stands out due to the high level of chromosomal anomalies, especially mosaicism that arises prior to global embryonic genome activation. Molecular cytogenetic studies show that an average of 60% of in vitro derived embryos have at least one aneuploid cell by the time they are 3 days old. However, comprehensive studies of the chromosome content of individual cells have revealed that 25% of these embryos have no aneuploid cells, a fact that sits well with the knowledge that at most 1 in 5 have the capacity to implant. The evidence is that extensive mosaicism, affecting several chromosomes, interferes with development to a greater extent than does uniform aneuploidy. Follow-up studies on embryos after pre-implantation genetic aneuploidy screening indicate that the frequency of meiotic errors varies according to the referral reason, with the highest frequency being recorded for the recurrent miscarriage category and the lowest in the repeated implantation failure group where younger women have a good response to ovarian stimulation. The exceptionally high incidence of pre- and post-zygotic chromosomal anomalies seen in early human embryos is thus the product of several mechanisms. Firstly, the error-prone cell cycle during the embryonic cleavage stage and secondly, parental susceptibility to meiotic and mitotic chromosomal instability together with their general genetic background.     

Epigenetic inactivation of the <Emphasis Type="Italic">RB1</Emphasis> gene as a factor of genomic instability: A possible contribution to etiology of chromosomal mosaicism during human embryo development

The methylation status of the cell cycle control gene RB1 has been studied in placental tissues of spontaneous abortions of the first trimester of pregnancy with mosaic variants of numerical chromosomal abnormalities verified by a molecular cytogenetic examination. Aberrant methylation of the gene promoter region has been revealed for the first time in 20% of embryos with chromosomal mosaicism that died in utero. A maximum frequency of epimutations was recorded in a group of embryos with a low level of abnormal cells for which mitotic errors are most likely to determine the formation of mosaic aneuploidy in primary euploid zygotes. It has been suggested that aberrant epigenetic genomic modifications at early stages of human embryonic development can be one of the mechanisms promoting genomic instability realized in the form of mosaic abnormalities of the karyotype that are incompatible with the normal course of embryogenesis.     

Regional and developmental characteristics of human embryo mosaicism revealed by single cell sequencing

Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several “complementary” copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.     

Evidence for high frequency of chromosomal mosaicism in spontaneous abortions revealed by interphase FISH analysis.

Numerical chromosomal imbalances are a common feature of spontaneous abortions. However, the incidence of mosaic forms of chromosomal abnormalities has not been evaluated. We have applied interphase multicolor fluorescence in situ hybridization using original DNA probes for chromosomes 1, 9, 13, 14, 15, 16, 18, 21, 22, X, and Y to study chromosomal abnormalities in 148 specimens of spontaneous abortions. We have detected chromosomal abnormalities in 89/148 (60.1%) of specimens. Among them, aneuploidy was detected in 74 samples (83.1%). In the remaining samples, polyploidy was detected. The mosaic forms of chromosome abnormality, including autosomal and sex chromosomal aneuploidies and polyploidy (31 and 12 cases, respectively), were observed in 43/89 (48.3%) of specimens. The most frequent mosaic form of aneuploidy was related to chromosome X (19 cases). The frequency of mosaic forms of chromosomal abnormalities in samples with male chromosomal complement was 50% (16/32 chromosomally abnormal), and in samples with female chromosomal complement, it was 47.4% (27/57 chromosomally abnormal). The present study demonstrates that the postzygotic or mitotic errors leading to chromosomal mosaicism in spontaneous abortions are more frequent than previously suspected. Chromosomal mosaicism may contribute significantly to both pregnancy complications and spontaneous fetal loss.     

Mechanisms of aneuploidy induction in human oogenesis and early embryogenesis

The mechanisms of aneuploidy induction in human oogenesis mainly involve nondisjunction arising during the first and second meiotic divisions. Nondisjunction equally affects both whole chromosomes and chromatids, in the latter case it is facilitated by "predivision" or precocious centromere division. Karyotyping and CGH studies show an excess of hypohaploidy, which is confirmed in studies of preimplantation embryos, providing evidence in favour of anaphase lag as a mechanism. Preferential involvement of the smaller autosomes has been clearly shown but the largest chromosomes are also abnormal in many cases. Overall, the rate of chromosomal imbalance in oocytes from women aged between 30 and 35 has been estimated at 11% from recent karyotyping data but accruing CGH results suggest that the true figure should be considerably higher. Clear evidence has been obtained in favour of germinal or gonadal mosaicism as a predisposing factor. Constitutional aneuploidy in embryos is most frequent for chromosomes 22, 16, 21 and 15; least frequently involved are chromosomes 14, X and Y, and 6. However, embryos of women under 37 are far more likely to be affected by mosaic aneuploidy, which is present in over 50% of 3-day-old embryos. There are two main types, diploid/aneuploid and chaotic mosaics. Chaotic mosaics arise independently of maternal age and may be related to centrosome anomalies and hence of male origin. Aneuploid mosaics most commonly arise by chromosome loss, followed by chromosome gain and least frequently by mitotic nondisjunction. All may be related to maternal age as well as to lack of specific gene products in the embryo. Partial aneuploidy as a result of chromosome breakage affects a minimum of 10% of embryos.     

Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH

Multicolour fluorescence in situ hybridisation (FISH) analysis of interphase nuclei in cleavage stage human embryos has highlighted a high incidence of postzygotic chromosomal mosaicism, including both aneuploid and ploidy mosaicism. Indeed, some embryos appear to have a chaotic chromosomal complement in a majority of nuclei, suggesting that cell cycle checkpoints may not operate in early cleavage. Most of these studies, however, have only analysed a limited number of chromosomes (3-5), making it difficult to distinguish FISH artefacts from true aneuploidy. We now report analysis of 11 chromosomes in five sequential hybridisations with standard combinations of two or three probes and minimal loss of hybridisation efficiency. Analysis of a series of arrested human embryos revealed a generally consistent pattern of hybridisation on which was superimposed frequent deletion of one or both chromosomes of a specific pair in two or more nuclei indicating a clonal origin and continued cleavage following chromosome loss. With a binucleate cell in a predominantly triploid XXX embryo, the two nuclei remained attached during preparation and the chaotic diploid/triphoid status of every chromosome analysed was the same for each nucleus. Furthermore, in each hybridisation the signals were distributed as a mirror-image about the plane of attachment, indicating premature decondensation during anaphase consistent with a lack of checkpoint control.     

Identification of Chromosomal Errors in Human Preimplantation Embryos with Oligonucleotide DNA Microarray

A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.     

The origin and impact of embryonic aneuploidy

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.     

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.     

Estimation of the mehylation status of the promoter region of the cell cycle control gene <Emphasis Type="Italic">P14ARF</Emphasis> in placental tissues of spontaneous abortions with chromosomal mosaicism

The methylation status of the promoter region of the cell cycle gene P14ARF was studied in the extraembryonic mesoderm and in the cytotrophoblast of 46 human spontaneous abortions with chromosomal mosaicism. Aberrant methylation of alleles of this gene was revealed for the first time in placental tissues of 9% of embryos. The identified epimutations were found to be characteristic of embryos with aneuploid cell clones of postzygotic origin. It is suggested that epigenetic inactivation of loci responsible for the regulation of cell division and for segregation of chromosomes is associated with the origin of mosaic forms of the karyotype at early stages of human embryonic development.     

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

A Euploid Line of Human Embryonic Stem Cells Derived from a 43,XX,dup(9q),+12,-14,-15,-18,-21 Embryo

Aneuploid embryos diagnosed by FISH-based preimplantation genetic screening (PGS) have been shown to yield euploid lines of human embryonic stem cells (hESCs) with a relatively high frequency. Given that the diagnostic procedure is usually based on the analysis of 1–2 blastomeres of 5 to 10-cell cleavage-stage embryos, mosaicism has been a likely explanation for the phenomena. However, FISH-based PGS can have a significant rate of misdiagnosis, and therefore some of those lines may have been derived from euploid embryos misdiagnosed as aneuploid. More recently, coupling of trophectoderm (TE) biopsy at the blastocyst stage and array-CGH lead to a more informative form of PGS. Here we describe the establishment of a new line of hESCs from an embryo with a 43,XX,dup(9q),+12,-14,-15,-18,-21 chromosomal content based on array-CGH of TE biopsy. We show that, despite the complex chromosomal abnormality, the corresponding hESC line BR-6 is euploid (46,XX). Single nucleotide polymorphism analysis showed that the embryo´s missing chromosomes were not duplicated in BR-6, suggesting the existence of extensive mosaicism in the TE lineage.     

Chromosomal mosaicism in spontaneous abortions: Analysis of 650 cases

It is known that up to 50% spontaneous abortions (SA) in the first trimester of pregnancy are associated with chromosomal abnormalities. We studied mosaic forms of chromosomal abnormalities in 650 SA specimens using interphase MFISH and DNA probes for chromosomes 1, 9, 13/21, 14/22, 15, 16, 18, X, and Y. Numerical chromosomal abnormalities were discovered in 58.2% (378 cases). They contained combined chromosomal abnormalities (aneuploidy of several chromosomes or aneuploidy in combination with polyploidy in the same specimen) in 7.7% (29 cases) or 4.5% of the entire SA sample; autosomal trisomy, in 45% (18.2% in chromosome 16, 8.9% in chromosomes 14/22, 7.9% in chromosomes 13/21, 3.1% in chromosome 18, and 1.4% in chromosome 9). Chromosome X aneuploidy was found in 27% cases, among which 9.6% represented chromosome X monosomy. Polyploidy was observed in 22.9% cases. In 5.1% cases, we observed mosaic form of autosomal monosomy. Among the SA cases with chromosomal abnormalities mosaicism was observed in 50.3% (∼ 25% of the entire SA sample). The results of the present study indicate that significant amount of chromosomal abnormalities in SA cells are associated with disturbances in mitotic chromosome separation, which represents the most common cause of intrauterine fetal death. It was also shown that original collection of DNA probes and the technique of interphase MFISH could be useful for detection of chromosomal mosaicism in prenatal cell specimens.     

Tetrasomy 9p mosaicism associated with a normal phenotype in two cases

Tetrasomy 9p is a rare chromosomal syndrome and about 30% of known cases exhibit mosaicism. Approximately 50 of the reported cases with tetrasomy 9p mosaicism show a characteristic facial appearance, growth failure, and developmental delay. However, 3 patients with mosaicism for isochromosome 9p and a normal phenotype have also been reported. We report 2 additional cases of clinically normal young females with tetrasomy 9p mosaicism, one of whom also exhibited X chromosome aneuploidy mosaicism leading to an overall of 6 different cell lines. STR analysis performed on this complex mosaic case indicated that the extra isochromosome was of maternal origin while the X chromosome aneuploidy was of paternal origin, indicating a postzygotic event.     

Mosaic aneuploidy in early fetal losses

Chromosomal mosaicism is the presence of 2 or more cell lines with different karyotypes in the same individual. Mosaic karyotypes are a remarkable feature of early stages of human embryo development. They result from mitotic errors in chromosome segregation and demonstrate the clearest example of somatic mutagenesis in human beings. This review is devoted to the classification of chromosomal mosaicism and the analysis of its underlying mechanisms, incidence and phenotypic effects during embryo development. A model for tissue-specific aneuploid cell line compartmentalization in spontaneous abortions is introduced.     

Sex ratio trajectory in mouse

Skewing of the sex ratio towards males occurs in humans. The possible explanation for excess male births could be a preference for Y-bearing sperm at fertilization and/or selective elimination of female embryos during pregnancy. In this study, we have tested the sex ratio in the preimplantation embryo (2–3 cells stage/closest possible primary sex ratio), the post-implantation embryo (day E7.5), and at birth (secondary sex ratio) on a homogenous (genetic, environmental, and dietary) population of mice to ascertain the biological reason i.e., male preference at fertilization or female elimination during pregnancy or both. Primary sex ratio on early preimplantation embryos (2–3 cells stage) was studied on 598 embryos and secondary sex ratio (at birth) on 721 pups using PCR-based sexing (both X & Y chromosome-specific) besides sex ratio of 80 post-implantation embryos (day E7.5). We have also investigated whether the fat content (high & low) of the diet affects the sex ratio. We observed a skewed sex ratio (more female) in preimplantation embryos (0.436; 95 % CI 0.39, 0.48), and post-implantation embryos (0.462; 95 % CI 0.35, 0.57) but reverse skewing (more male) at birth (0.539; 95 % CI 0.5, 0.58). We also observed that high-fat diet promoted male sex ratio at birth (0.657; 95 % CI 0.57, 0.74) whereas a low-fat diet had the opposite effect (0.46; 95 % CI 0.36, 0.56) but no effect at fertilization (2–3 cells stage embryos). This indicates selective elimination of female embryo and fetus throughout pregnancy in mice, more so with a high-fat diet.     

To err (meiotically) is human: the genesis of human aneuploidy

Aneuploidy (trisomy or monosomy) is the most commonly identified chromosome abnormality in humans, occurring in at least 5% of all clinically recognized pregnancies. Most aneuploid conceptuses perish in utero, which makes this the leading genetic cause of pregnancy loss. However, some aneuploid fetuses survive to term and, as a class, aneuploidy is the most common known cause of mental retardation. Despite the devastating clinical consequences of aneuploidy, relatively little is known of how trisomy and monosomy originate in humans. However, recent molecular and cytogenetic approaches are now beginning to shed light on the non-disjunctional processes that lead to aneuploidy.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

Dynamic karyotype,dynamic proteome: buffering the effects of aneuploidy

Despite its ubiquity in cancer, link with other pathologies, and role in promoting adaptive evolution, the effects of aneuploidy or imbalanced chromosomal content on cellular physiology have remained incompletely characterized. Significantly, it appears that the detrimental as well as beneficial effects of aneuploidy are due to the altered gene expression elicited by the aneuploid state. In this review we examine the correlation between chromosome copy number changes and gene expression in aneuploid cells. We discuss the primary effects of aneuploidy on gene expression and describe the cellular response to altered mRNA and protein levels. Moreover, we consider compensatory mechanisms that may ameliorate imbalanced gene expression and restore protein homeostasis in aneuploid cells. Finally, we propose a novel hypothesis to explain the hitherto enigmatic abundance compensation of proteins encoded on supernumerary chromosomes.