Genesis Of Aneuploidy Research Articles

Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential.

Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy.

TroX: a new method to learn about the genesis of aneuploidy from trisomic products of conception.

An estimated 10-30% of clinically recognized conceptions are aneuploid, leading to spontaneous miscarriages, in vitro fertilization failures and, when viable, severe developmental disabilities. With the ongoing reduction in the cost of genotyping and DNA sequencing, the use of high-density single nucleotide polymorphism (SNP) markers for clinical diagnosis of aneuploidy and biomedical research into its causes is becoming common practice. A reliable, flexible and computationally feasible method for inferring the sources of aneuploidy is thus crucial. We propose a new method, TroX, for analyzing human trisomy data using high density SNP markers from a trisomic individual or product of conception and one parent. Using a hidden Markov model, we infer the stage of the meiotic error (I or II) and the individual in which non-disjunction event occurred, as well as the crossover locations on the trisomic chromosome. A novel and important feature of the method is its reliance on data from the proband and only one parent, reducing the experimental cost by a third and enabling a larger set of data to be used. We evaluate our method by applying it to simulated trio data as well as to genotype data for 282 trios that include a child trisomic for chromosome 21. The analyses show the method to be highly reliable even when data from only one parent are available. With the increasing availability of DNA samples from mother and fetus, application of approaches such as ours should yield unprecedented insights into the genetic risk factors for aneuploidy. An R package implementing TroX is available for download at http://przeworski.uchicago.edu/.

Maternal-Restraint Stress Increases Oocyte Aneuploidy by Impairing Metaphase I Spindle Assembly and Reducing Spindle Assembly Checkpoint Proteins in Mice1

Studies in both humans and animals suggest detrimental effects of psychological stress on reproduction. Although our recent study shows that maternal-restraint stress diminishes oocyte developmental potential, the mechanism behind this effect is unknown. This prompted us to study the potential role of maternal-restraint stress in the genesis of aneuploidy during meiosis I. At 24 h after equine chorionic gonadotropin injection, mice were subjected to restraint stress for 24 h. After the restraint, some mice were killed to recover immature oocytes for in vitro maturation, while others were injected with human chorionic gonadotropin to recover in vivo matured oocytes. Analysis on chromosome complements of both mature oocytes and parthenotes confirmed that maternal restraint increased aneuploidy in both in vivo and in vitro matured oocytes and that the percentage of aneuploid oocytes were three times higher in the earlier matured oocytes than in the later matured ones. Further observations indicated that maternal restraint 1) impaired metaphase I (MI) spindle assembly while inhibiting MAPK activities, 2) accelerated progression of anaphase I while down-regulating the expression of spindle assembly checkpoint (SAC) proteins, and 3) induced intraoocyte oxidative stress. The following possible model was proposed to explain the results. Maternal-restraint stress increased oocyte aneuploidy by impairing MI spindle assembly and decreasing the SAC. Whereas abnormal spindles would affect centromere attachments, a reduction in SAC would accelerate the anaphase I progression. Failure of centromere attachment, together with the hastened anaphase, would result in nondisjunction of the unattached chromosomes. Furthermore, maternal-restraint stress might also impair spindle assembly and SAC function by inducing intraoocyte oxidative stress, which would then reduce MAPK activity, a critical regulator of microtubule assembly and the establishment and maintenance of the SAC during oocyte maturation.

Meiotic errors in human oogenesis and spermatogenesis

Chromosome anomalies are extraordinarily common in human gametes, with approximately 21% of oocytes and 9% of spermatozoa abnormal. The types of abnormalities are quite different since most abnormal oocytes are aneuploid, whereas the majority of abnormalities in spermatozoa are structural. Chromosomes 21 and 22 (the smallest chromosomes) are over-represented in aneuploid gametes in both oocytes and sperm. Chromosome 16 is also frequently observed in aneuploid oocytes, whereas the sex chromosomes are particularly predisposed to non-disjunction in human sperm. Maternal age is clearly the most significant factor in the aetiology of aneuploidy; theories about the cause of the maternal age effect are discussed. Paternal age does not have a dramatic effect on the frequency of aneuploid sperm; there is some evidence for a modest increase in the frequency of sex chromosomal aneuploidy. Meiotic recombination has a significant effect on the genesis of aneuploidy in both females and males. New techniques, which allow the analysis of recombination along the synaptonemal complex, have yielded interesting new information in healthy and infertile individuals. There is a link between infertility and the genesis of chromosome abnormalities. Future studies will unravel more of the underlying causal factors.

Sequence of centromere separation: effect of 5-azacytidine-induced epigenetic alteration.

The factors which control the sequential separation of the various chromosomes in a genome at the meta-anaphase junction are not well understood. In genomes in which separation is correlated with the quantity of pericentric heterochromatin one factor appears to be the epigenetic nature, namely condensation, of pericentric heterochromatin. When we induced decondensation of pericentric heterochromatin in mouse cells with 10(-6), 4x 10(-6) and 6x10(-6) M 5-azacytidine (5-AC) for 8 h, it resulted in alteration of the sequence of centromere separation. The centromeres which lacked pericentric heterochromatin appeared not to have been affected because there could not be an epigenetic alteration induced by 5-AC. The major effect was on chromosomes with the largest quantity of pericentric heterochromatin. These chromosomes separated at significantly higher frequency than in the untreated population. We also treated human cells, in which separation does not depend upon the quantity of heterochromatin, with 2x10(-5) and 6x10(-6) M 5-AC for 5 and 8 h. Compared with the control, 5-AC treatment resulted in an increased frequency of separated centromeres of acrocentric chromosomes in relation to those of non-acrocentric chromosomes. In the control the acrocentric chromosomes are the last to separate; in the treated population there was almost random separation of the two types of chromosomes. This epigenetic alteration might be another factor which results in genesis of aneuploidy.

Chromosomes lacking kinetochore proteins: their meta-anaphase location suggests potential malsegregation.

We have previously reported that a rare chromosome may not carry the kinetochore protein complex--the CENtromere Proteins or CENPs. These chromosomes should not bind to spindle microtubules and, hence, should be found peripheral to the meta-anaphase arrangement exhibited by the chromosomes which do carry CENPs. This communication shows that this actually is the case. When 3T3 mouse cells were not treated with colcemid or hypotonic, the kinetochore-lacking (K-) as well as kinetochore-bearing (K+) chromosomes were found off the spindle zone. When the spindle is disrupted with mild hypotonic treatment or by colcemid, the frequency of K- chromosomes remains unchanged. However, even mild disruption of the spindle with hypotonic treatment increases the frequency of off-lying K+ chromosomes significantly. These data indicate that K- chromosomes do not bind to the spindle and, hence, are a factor in the genesis of aneuploidy. A considerable proportion of K- chromosomes carry the putative centromere DNA indicating that these are not acentric fragments. Since the CREST serum used recognizes all essential kinetochore proteins, the K- centromeres must also lack all essential CENPs.

Are dislocated metaphase chromosomes and loss of kinetochores involved in the genesis of aneuploidy?

Are dislocated metaphase chromosomes and loss of kinetochores involved in the genesis of aneuploidy?

Kinetochore proteins, peripheral location of chromosomes and nuclear budding: another look at the genesis of aneuploidy

Some chromosomes in the rat do not have detectable levels of kinetochore proteins as determined by antikinetochore antibody, which may constitute a mechanism for the genesis of aneuploidy. An analysis of three mouse cell lines for the presence of kinetochore proteins showed that some chromosomes lacked these proteins, i.e. are akinetochoric by this criterion. This phenomenon may contribute to the variability of chromosome number that is characteristic of such lines. In some cells there appears to be en masse detachment of kinetochores. Several kinetochore-bearing chromosomes were also observed to be located peripherally to the spindle. These might be associated with the genesis of aneuploidy. In one cell line an apparently new phenomenon of nuclear budding was observed. In this case, one to several chromosomes appear to be pushed out of the nucleus in bud-like structures. These were surrounded by a nuclear membrane and appeared to detach from the main nucleus. Perhaps these structures eventually break off as micronuclei and, hence, would also be associated with the genesis of aneuploidy.