Frequency and distribution of aneuploidy in human gametes: differences as a function of sex]

The frequency and the distribution of aneuploidies were analysed in both spermatozoa and mature oocyte. The present study has pooled 13,975 human sperm chromosome complements and 1,897 oocyte chromosome complements examined to date. The overall frequency of aneuploidy is 10% in spermatozoa and 22.4% in oocytes. Human sperm is characterized by a significant excess of hypo-haploidies and an equitable distribution of aneuploidies among all chromosome groups, whereas mature oocytes display an equal ratio of hypo-haploidies: hyper-haploidies and a high variability in the distribution of non-disjunctions; in the A, B, C and especially in D and G groups, there is a significant difference between the observed and estimated rates of non-disjunction and the frequencies expected from an equal partitioning of non-disjunctions among all chromosomes. This indicates that non-disjunction is not a random event in female meiosis, and consequently that there are differences in the meiotic process between the sexes.     

Chromosomal analysis of unfertilized human oocytes

Unfertilized human oocytes were obtained from women in an in-vitro fertilization programme. The women had a mean age of 29.4 years (range 24-35 years). Chromosomal complements could be analysed in 50 oocytes. Q-banding of the chromosomes facilitated identification of individual chromosomes: 34 oocytes (68%) had the normal haploid chromosomal complement, 14 complements were hypohaploid (28%), 1 complement was hyperhaploid (2%) and 2 had structural abnormalities (4%). (One oocyte had numerical and structural abnormalities). The 16 abnormal oocytes were obtained from 15 different women. A conservative estimate of aneuploidy in this sample is 4%; however, the frequency of aneuploidy may be higher if there is a predisposition to chromosome loss during oogenesis. This study provides information on the largest series of karyotyped unfertilized human oocytes published to date.     

Enhanced polarizing microscopy as a new tool in aneuploidy research in oocytes

Chromosomal non-disjunction in female meiosis gives rise to reduced fertility and trisomy in humans. Human oocytes, especially from aged women, appear especially susceptible to non-disjunction. The oocyte spindle is crucial for high fidelity of chromosome segregation at meiotic divisions, and alterations in spindle morphology are therefore indicators of adverse conditions during oocyte development that may result in meiotic aneuploidy. In the past, oocytes had to be fixed for spindle analysis, precluding direct non-invasive identification of aneugens and adverse maturation conditions that affect spindle integrity and chromosome behaviour. Aneuploidy research for detection of spindle aberrations was therefore mainly focused on in vivo or in vitro exposed, fixed animal oocytes or cytogenetic analysis of spread oocytes. Orientation independent enhanced polarizing microscopy with nearly circularly polarized light and electronically controlled liquid crystal compensator optics is a new tool to study spindle morphology non-invasively in vivo for qualitative as well as quantitative analysis. Image generation by polarization microscopy depends on the intrinsic optical properties of the spindle with its paracrystalline microtubule lattice. When polarized light passes through such a lattice it induces a splitting of the beam and shift in the plane of vibration and retardation of light (termed birefringence and retardance). Studies of animal oocytes and follicle-cell denuded human oocytes fertilized by intracytoplasmic sperm injection for assisted conception have demonstrated the safety and efficacy of enhanced polarization microscopy. The method can be employed in aneuploidy research for non-invasive dose-response studies to detect spindle aberrations, for instance, in combination with cytogenetic analysis. Due to the non-invasive nature of the technique it may be employed in routine analysis of human oocytes to assess risks by lifestyle factors, and occupational and adverse environmental exposures.     

Cytogenetic analysis of Q-banded pronuclear chromosomes in fertilized Syrian hamster eggs

The frequency and type of chromosome abnormalities were analyzed in 917 female pronuclei in Syrian hamster eggs fertilized by human sperm. Analysis at this stage allows detection of errors which have occurred during meiosis I and II. The chromosomes were Q-banded to identify individual chromosomes and detect subtle alterations. Thirty-three (3.6%) of the hamster egg complements were abnormal: 19 (2.1%) were hypohaploid, seven (0.76%) were hyperhaploid, two (0.2%) had double aneuploidy, and five (0.5%) had a structural chromosome abnormality. Since there were significantly more hypohaploid than hyperhaploid complements, a conservative estimate of aneuploidy can be derived by doubling the frequency of hyperhaploid complements. Thus a minimal estimate of aneuploidy (single, 1.5%, and double, 0.2%) is 1.7% and a minimal estimate of the total frequency of abnormalities is 2.2%. All chromosome groups were represented among the aneuploid complements suggesting that all chromosomes are susceptible to non-disjunction.     

Human oocyte chromosome analysis: complicated cases and major pitfalls

Human oocytes that remained unfertilized in programmes of assisted reproduction have been analysed cytogenetically for more than 20 years to assess the incidence of aneuploidy in female gametes. However, the results obtained so far are not indisputable as a consequence of difficulties in evaluating oocyte chromosome preparations. Because of the lack of guidelines, we decided to summarize for the first time, the possible pitfalls in human oocyte chromosome analysis. Therefore, we screened the material from our previous studies and compiled representative, complicated cases with recommendations for their cytogenetic classification. We point out that maturity and size of the oocyte are important parameters and that fixation artefacts, as well as the particular structure of oocyte chromosomes, may predispose one to misinterpretations. Moreover, phenomena related to oocyte activation and fertilization are illustrated and explained. This compilation may help to avoid major problems in future studies and contribute to a more precise, and uniform assessment of human oocyte chromosomes.     

Molecular cytogenetic study of Robertsonian translocation 13;14 and Down syndrome in a 3-year-old infant

We describe here a rare case of Robertsonian translocation 13;14 of maternal origin combined with regular trisomy 21 (46,XX,der(13;14)(q10;q10)mat,+21) with Down syndrome phenotype. Molecular cytogenetic studies allowed us to determine the maternal origin of additional chromosome 21 and the non-disjunction of chromosome 21 to occur in meiosis I. On the basis of data obtained we discuss the possible involvement of structural alterations of chromosomes 13 and 14 in the chromosome 21 non-disjunction.     

Comparison of chromosomal abnormalities in hamster egg and human sperm pronuclei

One thousand human sperm and hamster egg haploid karyotypes were analyzed at the pronuclear stage after in vitro penetration. The frequency of abnormalities in human sperm was 8.5%, with 5.2% aneuploidy and 3.3% structural abnormalities. The hamster egg complements had an abnormality rate of 3.8%, with 3.3% aneuploidy and 0.5% structural abnormalities. In both human and hamster complements, chromosome abnormalities were observed in all chromosome groups, demonstrating that all chromosomes are susceptible to nondisjunction, not just acrocentric or small chromosomes. There is an intriguing difference between the frequency of hyperhaploid and hypohaploid complements in human sperm and hamster eggs. In the human complements, 2.4% were hyperhaploid and 2.7% hypohaploid. This is very close to the theoretical 1 to 1 ratio expected from nondisjunction. The hamster egg complements had more hypohaploid (2.2%) than hyperhaploid (0.9%) complements, despite identical treatment. Higher rates of hypohaploidy are generally ascribed to artificial loss of chromosomes, but may in fact reflect a predisposition of oocytes to anaphase lag during meiosis. The frequency of abnormalities (both numerical and structural) is higher in human complements than in hamster. This may reflect an innate propensity for meiotic chromosome abnormalities in humans or may result from greater exposure of humans to mutagenic agents.     

The incidence of aneuploidy in human oocytes assessed by conventional cytogenetic analysis

Human oocytes failing to fertilize during assisted reproduction are an important source of information for assessing incidence and causal mechanisms of maternal aneuploidy. This review describes the techniques of conventional oocyte chromosome analysis and evaluates the results of 59 studies comprising a total of>10,000 female gametes. The mean rate of aneuploidy (hypohaploidy + hyperhaploidy) amounts to approximately 20%, but this incidence is reduced as soon as possible artifacts introduced by the fixation technique are taken into consideration. It is therefore concluded that a realistic value for numerical abnormalities arising during first meiotic division lies between 12 and 15%. All chromosome groups are affected by aneuploidy but the actually observed frequencies exceed the expected frequencies in groups D, E, and G. Two aneuploidy-causing mechanisms have been identified in human oocytes: nondisjunction, resulting in the loss or gain of whole chromosomes, and predivision, resulting in the loss or gain of single chromatids. A brief analysis including only aneuploid complements with one extra or missing chromosome/chromatid shows a slight increase in predivision (52.9%) compared with nondisjunction (47.1%). Finally, suggestions for future studies are given since, for instance, the presentation of results and the use of cytogenetic nomenclature have not been uniform.     

Sperm chromosome complements in a 47,XYY man

Summary Human sperm chromosomes from a 47,XYY male were examined using the direct method of sperm chromosome analysis with two modifications in the semen processing. A total of 75 sperm complements was karyotyped and all of these contained one sex chromosome. The percentages of X-and Y-bearing sperm were 53% and 47%, respectively. There were 10 sperm with autosomal chromosomal abnormalities. The frequencies of numerical (4.0%), structural (10.6%), and total (13.3%) abnormalities were not significantly different from the frequencies observed in normal donors in our laboratory. Our results do not support the suggestion that XYY males have an increased risk of aneuploid progeny as a result of secondary non-disjunction or interchromosomal effects. They do support the hypothesis that one Y chromosome is eliminated in the germ cells of XYY males. However since our study provides the first information on sperm chromosomes in an XYY male, further studies on other XYY men are required.     

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.     

Frequency and distribution of chromosome abnormalities in human spermatozoa

This study reviews the frequency and distribution of numerical and structural chromosomal abnormalities in spermatozoa from normal men obtained by the human-hamster system and by multicolor-FISH analysis on decondensed sperm nuclei. Results from large sperm karyotyping series analyzed by chromosome banding techniques and results from multicolor FISH in sperm nuclei (of at least 10(4) spermatozoa per donor and per probe) were reviewed in order to establish baseline values of the sperm chromosome abnormalities in normal men. In karyotyping studies, the mean disomy frequency in human sperm is 0.03% for each of the autosomes, and 0.11% for the sex chromosomes, lower than those reported in sperm nuclei by FISH studies using a similar methodology (0.09% and 0.26%, respectively). Both types of studies coincide in that chromosome 21 and sex chromosomes have a greater tendency to suffer segregation errors than the rest of the autosomes. The mean incidence of diploidy, only available from multicolor FISH in sperm nuclei, is 0.19%. Inter-donor differences observed for disomy and diploidy frequencies among FISH studies of decondensed sperm nuclei using a similar methodology could reflect real differences among normal men, but they could also reflect the subjective application of the scoring criteria among laboratories. The mean frequency of structural aberrations in sperm karyotypes is 6.6%, including all chromosome types of abnormalities. Chromosome 9 shows a high susceptibility to be broken and 50% of the breakpoints are located in 9q, between the centromere and the 9qh+ region. Structural chromosome aberrations for chromosomes 1 and 9 have also been analyzed in human sperm nuclei by multicolor FISH. Unfortunately, this assay does not allow to determine the specific type of structural aberrations observed in sperm nuclei. An association between advancing donor age and increased frequency of numerical and structural chromosome abnormalities has been reported in spermatozoa of normal men.     

Does human oocyte cryopreservation affect equally on embryo chromosome aneuploidy?

Oocytes cryopreservation as an important part of assisted reproductive technologies, which should ensure after warming not only intact oocyte morphological characteristics, but also their genetic apparatus stability. However, the meiotic spindle is very sensitive to the temperature fluctuations that can lead to unequal chromosome segregation during meiosis and as a consequence can cause embryo aneuploidy after oocyte fertilization. The aim of the study was to estimate the oocytes cryopreservation impact on human embryo chromosome aneuploidy. It has been shown that fertilization rate of the cryopreserved oocytes did not differ from fresh ones (83.1% vs 84% respectively). The number of blastocysts obtained from cryopreserved oocytes was less than that obtained from fresh oocytes, however, their morphological characteristics were better if compared the fresh oocytes. Our results showed different cryopreservation impact on aneuploidy rates of certain chromosomes in embryos obtained from cryopreserved oocytes. They had an increased aneuploidy of chromosome 13 and a decreased nondisjunction of chromosome 18 and sex chromosomes.     

Cytogenetic analysis of unfertilized human oocytes

The results of morpho-functional and cytogenetic analyses of 341 oocytes unfertilized in the course of extracorporal fertilization programme are presented. The causes of the "failure" during in vitro fertilization of the oocytes are discussed. Relation of the frequency of oocyte chromosome abnormalities (40.2%) on the patient age and cell maturity has been shown.     

Mechanisms of whole chromosome gains in tumors--many answers to a simple question

Whole chromosome gain is the most common type of gross genomic abnormality observed in human tumors. It is particularly frequent in lympho-haematopoietic and embryonic neoplasms, where trisomies and tetrasomies are typically present together with few or no other cytogenetic imbalances, resulting in hyperdiploid chromosome numbers. Despite the high prevalence of whole chromosome gains in neoplastic cells, their mechanism of origin remains disputed. Here, 4 potential models for the generation of whole chromosome gains are reviewed: (1) loss of chromosomes from the tetraploid level, (2) sequential sister chromatid non-disjunction, (3) multipolar mitosis coupled to sister chromatid non-disjunction, and (4) multipolar mitosis coupled to incomplete cytokinesis. Each of these mechanisms may in theory result in the generation of hyperdiploid neoplastic clones, but none of them were single-handedly able to reproduce the scenario of chromosome copy number alterations in tumors when cell populations resulting from these models were simulated in silico and compared to published cytogenetic data. To develop models for the generation of whole chromosome gains further, it is critical to improve our knowledge of the principles of clonal selection in tumors and of the baseline rate of chromosome segregation errors in human cells. To illustrate this, a model combining multipolar mitosis coupled to incomplete cytokinesis with a low rate of baseline sister chromatid non-disjunction was shown readily to reproduce copy number distributions in hyperdiploid karyotypes from human tumors.     

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.     

Effect of maternal age on the frequency of cytogenetic abnormalities in human oocytes

The cytogenetic investigation of human oocytes was initiated in the Sixties, and for the last four decades, this field of research has never stopped progressing as new technologies appear. Numerous karyotyping studies and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes, but also displaying a great variability in results, which may be essentially attributable to the technical limitations of these in situ methods when applied to human oocytes. Essentially, the most relevant analyses have led to the estimate that 15-20% of human oocytes display chromosome abnormalities, and they have emphasized the implication of both whole chromosome nondisjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidies has also been investigated in human oocytes. Most previous studies have failed to confirm any relationship between maternal age and aneuploidy frequency in human oocytes, whereas the more recent reports based on large samples of oocytes or polar bodies have provided evidence for a direct correlation between increased aneuploidy frequency and advanced maternal age, and have clarified the contribution of the various types of malsegregation in the maternal age-dependent aneuploidies.     

Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities.     

Chromosome abnormalities in the human oocyte

Aneuploidy is the most commonly occurring type of chromosome abnormality and the most significant clinically. It arises mostly due to segregation errors taking place during female meiosis and is also closely associated with advancing maternal age. Two main aneuploidy-causing mechanisms have been described: the first involves the non-disjunction of entire chromosomes and can take place during both meiotic divisions, whereas the second involves the premature division of a chromosome into its 2 sister chromatids, followed by their random segregation, upon completion of meiosis I. To elucidate the causal mechanisms of maternally derived aneuploidy and the manner with which they affect the 2 meiotic divisions, a large number of oocytes and their corresponding polar bodies have been examined. Various classical and molecular cytogenetic methods have been employed for this purpose, and valuable data have been obtained. Moreover, research into the gene expression patterns of oocytes according to maturity, maternal age, and chromosome status has provided a unique insight into the complex nature of the biological processes and genetic pathways regulating female meiosis. Findings obtained from the cytogenetic and molecular analysis of oocytes will be reviewed in this article.     

Non-disjunction of chromosome 21, alphoid DNA variation, and sociogenetic features of Down syndrome

The analysis of non-disjunction of chromosome 21 and alphoid DNA variation by using cytogenetic and molecular cytogenetic techniques (quantitative fluorescence in situ hybridization) in 74 nuclear families was performed. The establishment of possible correlation between alphoid DNA variation, parental age, environmental effects, and non-disjunction of chromosome 21 was made. The efficiency of techniques applied was found to be 92% (68 from 74 cases). Maternal non-disjunction wasfound in 58 cases (86%) and paternal non-disjunction - in 7 cases (10%). Post-zygotic mitotic non-disjunction was determined in 2 cases (3%) and one case was associated with Robertsonian translocation 46,XX,der(21;21)(q10;q10), +21. Maternal meiosis I errors were found in 43 cases (64%) and maternal meiosis II errors--in 15 cases (22%). Paternal meiosis I errors occurred in 2 cases (3%) and paternal meiosis I errors--in 5 cases (7%). The lack of the correlation between alphoid DNA variation and non-disjunction of chromosome 21 was established. Sociogenetic analysis revealed the association of intensive drug therapy of infectious diseases during the periconceptual period and maternal meiotic non-disjunction of chromosome 21. The correlation between non-disjunction of chromosome 21 and increased parental age as well as exposure to irradiation, alcohol, tobacco, mutagenic substances was not found. The possible relevance of data obtained to the subsequent studies of chromosome 21 non-disjunction is discussed.     

Imprint switching for non-random X-chromosome inactivation during mouse oocyte growth

In mammals, X-chromosome inactivation occurs in all female cells, leaving only a single active X chromosome. This serves to equalise the dosage of X-linked genes in male and female cells. In the mouse, the paternally derived X chromosome (X(P)) is imprinted and preferentially inactivated in the extraembryonic tissues whereas in the embryonic tissues inactivation is random. To investigate how X(P) is chosen as an inactivated X chromosome in the extraembryonic cells, we have produced experimental embryos by serial nuclear transplantation from non-growing (ng) oocytes and fully grown (fg) oocytes, in which the X chromosomes are marked with (1) an X-linked lacZ reporter gene to assay X-chromosome activity, or (2) the Rb(X.9)6H translocation as a cytogenetic marker for studying replication timing. In the extraembryonic tissues of these ng/fg embryos, the maternal X chromosome (X(M)) derived from the ng oocyte was preferentially inactivated whereas that from the fg oocyte remained active. However, in the embryonic tissues, X inactivation was random. This suggests that (1) a maternal imprint is set on the X(M) during oocyte growth, (2) the maternal imprint serves to render the X(M) resistant to inactivation in the extraembryonic tissues and (3) the X(M) derived from an ng oocyte resembles a normal X(P).