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.     

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4–8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility.     

Correlation between preimplantation genetic diagnosis for chromosomal aneuploidies and the efficiency of establishing human ES cell lines

There are several sources from which human embryonic stem cell (hESC) lines can be generated: surplus embryos after in vitro fertilization procedures, one- and three-pronuclear zygotes, early arrested or highly fragmented embryos that have reached the blastocyst stage, or otherwise chromosomally or genetically abnormal embryos after preimplantation genetic diagnosis (PGD). We report on the efficiency of establishing hESC lines from blastocysts with proven meiotic or mitotic errors after sequential testing of both polar bodies and blastomere analysis on day 3. The success rate of establishing hESC lines originating from blastocysts carrying a meiotic error was as low as 2.4% and differed significantly from the success rate of establishing hESC lines originating from blastocysts with balanced meiotic errors (21.6%) or mitotic errors (after sequential testing (9.1%) and after blastomere testing alone (12.2%)). This suggests that it may be reasonable to apply sequential PGD prior to the initiation of hESC culture. Information about the karyotype may in the future help refine the methods and possibly improve the efficiency by which hESC lines are derived from embryos with prezygotic abnormalities. Additionally, it may in general prove very difficult to obtain abnormal hESC lines for scientific study from aneuploid PGD embryos, which will limit our ability to study the biological consequences of chromosomal abnormalities. Furthermore, the success rates for generating aneuploid cell lines originating from fertilized oocytes carrying a prezygotic nondisjunction error seem to mirror the miscarriage rates during pregnancy of embryos carrying such errors.     

The in vitro survival of human monosomies and trisomies as embryonic stem cells

Chromosomal aneuploidies are responsible for severe human genetic diseases. Aiming at creating models for such disorders, we have generated human embryonic stem cell (hESC) lines from pre-implantation genetic screened (PGS) embryos. The overall analysis of more than 400 aneuploid PGS embryos showed a similar risk of occurrence of monosomy or trisomy for any specific chromosome. However, the generation of hESCs from these embryos revealed a clear bias against monosomies in autosomes. Moreover, only specific trisomies showed a high chance of survival as hESC lines, enabling us to present another categorization of human aneuploidies. Our data suggest that chromosomal haploinsufficiency leads to lethality at very early stages of human development.     

Frequency and distribution of chromosome abnormalities in human oocytes

It was previously shown that more than half of the human oocytes obtained from IVF patients of advanced reproductive age are aneuploid, due to meiosis I and meiosis II errors. The present paper further confirms that 61.8% of the oocytes tested by fluorescent probes specific for chromosomes 13, 16, 18, 21 and 22 are abnormal, representing predominantly chromatid errors, which are the major source of aneuploidy in the resulting embryos. Almost half of the oocytes with meiosis I errors (49.3%) are prone to sequential meiosis II errors, which may lead to aneuploidy rescue in 30.8% of the cases. Half of the detected aneuploidies (49.8%) are of complex nature with involvement of two or more chromosomes, or the same chromosome in both meiotic divisions. The aneuploidy rates for individual chromosomes are different, with a higher prevalence of chromosome 21 and 22 errors. The origin of aneuploidy for the individual chromosomes is also not random, with chromosome 16 and 22 errors originating more frequently in meiosis II, and chromosome 18, 13 and 21 errors in meiosis I. There is an age dependence not only for the overall frequency of aneuploidies, but also for each chromosome error, aneuploidies originating from meiosis I, meiosis II, and both meiosis I and meiosis II errors, as well as for different types of aneuploidies. The data further suggest the practical relevance of oocyte aneuploidy testing for detection and avoidance from transfer of the embryos deriving from aneuploid oocytes, which should contribute significantly to the pregnancy outcomes of IVF patients of advanced reproduction age.     

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20-50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.     

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.     

Mouse emi1 has an essential function in mitotic progression during early embryogenesis

For successful mitotic entry and spindle assembly, mitosis-promoting factors are activated at the G(2)/M transition stage, followed by stimulation of the anaphase-promoting complex (APC), an E3 ubiquitin ligase, to direct the ordered destruction of several critical mitotic regulators. Given that inhibition of APC activity is important for preventing premature or improper ubiquitination and destruction of substrates, several modulators and their regulation mechanisms have been studied. Emi1, an early mitotic inhibitor, is one of these regulatory factors. Here we show, by analyzing Emi1-deficient embryos, that Emi1 is essential for precise mitotic progression during early embryogenesis. Emi1(-/-) embryos were found to be lethal due to a defect in preimplantation development. Cell proliferation appeared to be normal, but mitotic progression was severely defective during embryonic cleavage. Moreover, multipolar spindles and misaligned chromosomes were frequently observed in Emi1 mutant cells, possibly due to premature APC activation. Our results collectively suggest that the late prophase checkpoint function of Emi1 is essential for accurate mitotic progression and embryonic viability.     

Deficiency in the response to DNA double-strand breaks in mouse early preimplantation embryos

DNA double-strand breaks (DSBs) are caused by various environmental stresses, such as ionizing radiation and DNA-damaging agents. When DSBs occur, cell cycle checkpoint mechanisms function to stop the cell cycle until all DSBs are repaired; the phosphorylation of H2AX plays an important role in this process. Mouse preimplantation-stage embryos are hypersensitive to ionizing radiation, and X-irradiated mouse zygotes are arrested at the G2 phase of the first cell cycle. To investigate the mechanisms responding to DNA damage at G2 in mouse preimplantation embryos, we examined G2/M checkpoint and DNA repair mechanisms in these embryos. Most of the one- and two-cell embryos in which DSBs had been induced by gamma-irradiation underwent a delay in cleavage and ceased development before the blastocyst stage. In these embryos, phosphorylated H2AX (gamma-H2AX) was not detected in the one- or two-cell stages by immunocytochemistry, although it was detected after the two-cell stage during preimplantation development. These results suggest that the G2/M checkpoint and DNA repair mechanisms have insufficient function in one- and two-cell embryos, causing hypersensitivity to gamma-irradiation. In addition, phosphorylated ataxia telangiectasia mutated protein and DNA protein kinase catalytic subunits, which phosphorylate H2AX, were detected in the embryos at one- and two-cell stages, as well as at other preimplantation stages, suggesting that the absence of gamma-H2AX in one- and two-cell embryos depends on some factor(s) other than these kinases.     

Aneuploidy in the human blastocyst

Studies of human cleavage stage embryos, 3 days after fertilization of the oocyte, have revealed remarkably high levels of chromosome abnormality. In addition to meiotic errors derived from the gametes, principally the oocyte, mitotic errors occurring after fertilization are also common, leading to widespread chromosomal mosaicism. The prevalence of chromosome anomalies in embryos may explain the relatively poor fertility and fecundity in humans and the low success rates of assisted reproductive treatments (e.g., IVF). While much is known concerning the incidence of aneuploidy during the first 3 days following fertilization, it is only in the last couple of years that large numbers of embryos at the final stage of preimplantation development, the blastocyst stage, 5 days after fertilization, have been subjected to detailed analysis. Here we discuss the latest data from the comprehensive cytogenetic analysis of blastocysts. These findings indicate that the majority of selection against chromosome abnormalities does not occur until the time of implantation or shortly after, with aneuploidy typically affecting more than 50% of blastocysts. Additionally, clinical results presented suggest that screening of blastocyst stage embryos for chromosome abnormality, with preferential transfer to the uterus of those found to be euploid, may help to improve the success rates of assisted reproductive treatments.     

Mitochondria in early mammalian development

The role of mitochondria as central determinants of development competence of oocytes and preimplantation stage embryos is considered in the context of the diverse activities these organelles have in normal cell function. Stage- and cell-cycle-specific mitochondrial translocations and redistributions are described with respect to mechanisms of cytoplasmic remodeling that may establish domains of autonomous regulation of mitochondrial function and activity during early development. The functions of mitochondria as intracellular signaling elements, as regulators of signaling pathways, and oxygen sensors in differentiated cells are suggested to have similar capacities during mammalian oogenesis and early embryogenesis. Questions concerning the numerical size of the oocyte mitochondrial complement, the energy required to support normal preovulatory oogenesis and preimplantation embryogenesis, and the regulation of mitochondrial activity by extrinsic and intrinsic factors are addressed with respect the potential they may have for new investigational approaches to study the origin of the differential developmental competence of human oocytes and preimplantation stage embryos.     

Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients

We have used multicolour fluorescent in situ hybridisation (FISH) with DNA probes for chromosomes X, Y and 1 to analyse spare untransferred cleavage-stage embryos after preimplantation diagnosis to avoid X-linked disease. In total, 93 morphologically normal embryos were available from seven patients (six of proven fertility) who had undergone fourteen in vitro fertilisation (IVF) cycles. The chromosome patterns observed were classified into four groups; normal, abnormal (non-mosaic), mosaic and chaotic (uncontrolled division). Approximately half of the embryos were normal for the chromosomes tested. Two embryos only were aneuploid (non-mosaic) throughout but, after excluding those showing chaotic division, 30% were considered to be chromosomal mosaics. Of these, a minority had arisen because of mitotic non-disjunction or chromosome loss or gain, whereas the majority were ploidy mosaics, with haploidy being the most common. The occurrence of chaotically dividing embryos was strongly patient-related, i.e. some patients had ‘chaotic’ embryos in repeated cycles, whereas other patients were completely free of this type of anomaly. ‘Chaotic’ embryos are unlikely to progress beyond implantation. These findings have important implications both for routine IVF and preimplantation genetic diagnosis. Received: 18 October 1996 / Revised: 23 January 1997     

Amino acid transport regulation and early embryo development

Amino acids are essential components of media utilized to culture fertilized human eggs to the blastocyst stage in vitro. Use of such media has led to a significant increase in the proportion of embryos that implant upon transfer to the uterus and to a decrease in the number that need to be transferred to achieve pregnancy. Little is known about the mechanisms by which amino acids foster development of healthy human blastocysts. Indications are, however, that many of these mechanisms are the same in human and mouse embryos. Both essential and nonessential amino acid transport benefit preimplantation mouse embryo development, albeit at different stages. Nonessential amino acid transport improves development primarily during cleavage, whereas essential amino acid transport supports development of more viable embryos, especially subsequent to the eight-cell stage. This review discusses likely mechanisms for these beneficial effects.     

Chromosomal disorders and nuclear and cell destruction in cleaving human embryos

Three types of defects of preimplantation embryogenesis contribute to the developmental arrest of cleaving human embryos: blastomere fragmentation, abnormal nuclear status and chromosomal disorders. Data concerning the relation and succession of these abnormalities during first mitotic cycles of the human zygote are controversial and mainly empirical at present. In this study we have performed simultaneous evaluation of blastomere fragmentation, nuclear apoptotic changes and the ploidy of four chromosomes (1, 5, 19 and X or 18, 21, X and Y) in 193 human embryos. Another group of 28 embryos was subjected to TUNEL for confirmation of apoptosis in blastomere nuclei. Nuclei with apoptotic chromatin were seen in nearly 1/10 of blastomeres of embryos with good morphology and in more than 1/5 of blastomeres of embryos with more than 20% fragmentation. The correct number of investigated chromosomes was registered in 85.2% of successfully tested embryos. Chromatin apoptotic changes are the only limiting factor for the success of chromosomal FISH tests. Nearly 1/2 of embryos with at least one apoptotic nucleus were chromosomally abnormal. For the embryos that contain only normal nuclei, the rate of ploid normality was more than 89%. The rate of euploidy was higher (66%) in embryos with a significant degree of cell fragmentation. Moderate cell fragmentation was not related to significant increase of chromatin and chromosomal disorders. In a substantial portion of abnormal blastomeres, chromatin damage preceded cell fragmentation. Nuclear destruction in human blastomeres was illustrated by fluorograms of different stages of chromatin lesions.     

Expression of beta-galactosidase in preimplantation ovine and porcine embryos

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.