Overall, meiosis in the female is obviously more error-prone than in the male; this could result from the lack of checkpoint control at the metaphase—anaphase transition in female mammals, a suggestion for which some experimental evidence exists in the mouse LeMaire-Adkins et al. After fertilization in vitro on day 0, the embryo undergoes successive cleavage divisions, to consist of 6—10 cells by day 3 and maybe over by day 5, when blastocyst formation occurs, with separation of the inner cell mass and the trophectoderm.
The embryo proper is derived from the inner cell mass. Unlike mouse embryos, most human fertilized eggs in culture do not become blastocysts, but arrest in development at an earlier stage. Many studies employing routine karyotype analysis have been attempted on human pre-implantation embryos. At the cleavage stage, the embryo is analysed as a whole, after treatment to induce metaphase arrest. Nevertheless, it is readily apparent that there is a high frequency of chromosomal anomalies at this stage of development Jamieson et al. A technical advance was achieved by Clouston, who developed a way of obtaining good quality metaphases from blastocysts Clouston et al.
Further advance was driven by the need to develop pre-implantation genetic diagnosis PGD. By day 3 of development, it proved possible to remove one or two cells from the embryo and use these for molecular diagnosis Handyside, Metaphase preparation was not technically possible, but the application of FISH analysis to interphase nuclei rapidly became the method of choice when sexing the embryo to avoid X-linked disease.
Embryo Preimplantation Genetic Testing (PGT)
Very quickly it became apparent that chromosomal mosaicism, as well as aneuploidy, was rife in the day 3 embryo Delhanty et al. Embryos derived from both sets of patients showed the same types of abnormalities. They could be divided into different classes: completely diploid for the chromosomes examined; uniformly aneuploid; and mosaic.
An altogether different molecular approach was needed, namely comparative genomic hybridization CGH. Originally developed for use in cancer cytogenetics, when the tissue obtained cannot be readily induced to produce analysable metaphases, this is a DNA-based method employing FISH technology Kallionemi et al.
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Extracted DNA from the test Table 3. Data from Delhanty et al. The DNAs are then competitively co-hybridized to prepared chromosome spreads from a normal male. A variety of methods were investigated in detail and the most appropriate was determined to be degenerate oligonucleotide-primed polymerase chain reaction DOP-PCR. Twelve good-quality day 3 human embryos were then dissaggregated into single cells and the combination of DOP-PCR and CGH was then applied to obtain a comprehensive picture of the chromosome constitution of each individual cell.
The results were remarkable Wells and Delhanty, Most notable was that three of the embryos were completely euploid and had no chromosome imbalance. One was uniformly double aneuploid trisomy 21 and X monosomy , one had three of four cells with chromosome 1 monosomy. It seemed likely that, of the seven containing all or a majority of cells with abnormal chromosomes, four had a meiotic origin. In the same year, a comparable study was carried out in Australia, producing remarkably similar results Voullaire et al.
Table 3. Data from Wells and Delhanty 1 ; Voullaire et al. Embryo survival How important are these abnormalities, particularly mosaicism, for embryo survival? It is frequently stated that, since the embryo proper is derived from very few cells, the presence of a minority of chromosomally abnormal cells at the cleavage stage may not be important.
It is known that the presence of a normal cell line in the placenta greatly enhances the intrauterine survival of fetuses trisomic for chromosomes 13 and 18 Kalousek et al. Some information on the survival capabilities of different types of abnormalities is provided by allowing embryos diagnosed on the basis of a single cell analysed as chromosomally abnormal to grow on in culture.
Among the 50 blastocysts, 17 were aneuploid, 14 of which were trisomies. The three surviving monosomies were for chromosome 21 or the X. A reasonable conclusion could be that monosomies other than for chromosomes 21 or X were likely to be lethal prior to the blastocyst stage, and that extensive mosaicism slowed development considerably, making successful implantation unlikely.
The cause of high levels of chromosome abnormality in human embryos The incidence and type of post-zygotic errors leading to mosaicism that have been consistently observed in human cleavage stage embryos is totally unlike any observed in cultured somatic cells, suggesting that the mechanisms operating are peculiar to this stage of development.
In cancer cells or those transformed in culture, these checkpoints are often defective, allowing the sporadic accumulation of secondary chromosomal and other genetic defects. The human embryo is largely reliant on maternal transcripts until global activation of the embryonic genome at the 6—8 cell stage on day 3 Braude et al.
Unfortunately, it is not possible to carry out similar studies on embryos from natural conceptions, but it is of great interest that the classical observations of Hertig et al. If the frequent occurrence of chromosomal mosaicism and chaotically dividing embryos also applies to in vivo conceptions, this may explain the relatively poor rate of embryonic implantation in the human species.
Relative parental risks -- age, translocations, inversions, gonadal and germinal mosaics In the population as a whole, the most important risk factor for a chromosomally abnormal conception is advanced maternal age. Among recognized pregnancies, the main association is with trisomy; there is no increased risk with age for triploidy or monosomy X, the risk for which is in fact increased in young women. The causes of age-related aneuploidy have been debated for many years and numerous hypotheses have been proposed but, although some experimental evidence has been obtained, a clear understanding of the problem remains elusive.
This could involve growth of the immature follicles, defective assembly of the spindle, or failure of the paired chromosomes to align correctly upon the equator of the spindle. These couples require genetic counselling and in most cases appropriate prenatal diagnosis can be offered, ensuring that an ongoing pregnancy is chromosomally balanced.
However, a minority of such couples experience repeated early miscarriages or primary infertility; for this sub-group pre-implantation diagnosis with selective transfer of embryos is appropriate Conn et al. It is of considerable interest that follow-up studies on embryos from such cases that are not transferred due to chromosomal imbalance have shown exceptionally high levels of mosaicism in these couples with fertility problems Conn et al.
Preimplantation genetic testing (PGT) at a glance
A second group of couples at high risk of conceiving a chromosomally abnormal child are those in which one partner is a gonadal or germinal mosaic for a trisomic cell line. However, the evidence gained from preimplantation diagnosis in one such case suggests that the risks are in fact higher than would be expected from classical considerations Cozzi et al.
This is because the three copies of the chromosome may not associate in a trivalent in prophase of meioisis I, but as a bivalent and an unpaired monovalent single chromosome. The monovalent is then more likely to undergo premature separation into its constituent chromatids and these may segregate at random, producing additional unbalanced gametes. As explained earlier, germinal mosaicism may arise during mitosis in the premeiotic divisions of the germ cells and may affect one or several germ cells, again leading to a high-risk situation.
Since most cases of this syndrome are in fact born to women who are not of advanced age, it is clearly important to understand the possible causes leading to a high risk of abnormality, so that counselling and prenatal diagnosis can be offered where appropriate. References Angell, R. Angell, R.
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Saunders: Philadelphia, PA. Handyside, A. Plenum: New York; 75— Harper, J. H, Winston, R. Hartwell, L. Hassold, T. Hertig, A. Hunt, P. Iwarsson, E. Jacobs, P. Jamieson, M.
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Kallioniemi, A. Kalousek, D. Koehler, K. LeMaire-Adkins, R. Mahmood, R. Murray, A. Pellestor, F. Pujol, A. Sandalinas, M. Simopoulou, M.
Stavropoulos, D. Stevens, V. Update 3: — Volarcik, K. Voullaire, L. Wells, D. Nucleic Acids Res. White, M. Wilcox, A.