Chromosomal Chaos In Early Embryonic Development Linked To Cell Division Abnormalities

"Use of the diagnostic technique fluorescence in situ hybridisation (FISH), that utilises fluorescence probes to identify specific chromosomes, has previously revealed a large number of chromosomal abnormalities including 'chaos' in embryos before implantation", she said. "Up till now it was believed that these may be due to technical artefacts and not representative of the true status of the embryos. Our work has shown that this is not the case. In fact, we have identified a major pathway leading to chromosomal and nuclear abnormalities in the preimplantation embryo in vitro."

During cell division, spindle fibres align the 46 chromosomes along the middle of the cell and then forces are exerted that pull the chromosomes to opposite directions. A bi-polar spindle ensures that each of the two daughter cells receives a copy of each of the 46 chromosomes. Dr. Chatzimeletiou's team found that some spindles in cleavage and blastocyst stage embryos contain more than two poles (tripolar or tetraplolar), and in those it is impossible for the 46 chromosomes to be segregated equally to two daughter cells, as they would be pulled to three or more directions. "This in turn inevitably leads to chromosomal chaos", she said.

Dr. Chatzimeletiou and colleagues in Greece and Britain selected 185 cleavage to blastocyst stage human embryos and treated them with markers in order to visualise the microtubules, which are involved with motion activities within the cell, and spindles. All the blastocyst stage embryos had at least one normal spindle, but 23% of blastocysts had an abnormal spindle in addition to the normal ones. At the earlier cleavage to morula stages more abnormalities were seen (29% of spindles were abnormal vs 9% at the blastocyst). Chromosome loss was also seen in 16% of embryos and although this was frequently associated with abnormal spindle configurations, some otherwise normal spindles were also linked to chromosome loss.

"We think that multipolar spindles are due to abnormal replication of centrosomes (the organising centres and precursors of spindles in the cell)", said Dr. Chatzimeletiou. Each cell contains one centrosome that duplicates and moves to opposite poles in order to form a bipolar spindle. Multipolar spindles most likely arise in cells that have undergone one cycle of failed cytoplasmic division and have become either binucleate or tetraploid Those cells now contain 2 centrosomes instead of 1 from the previous division and if they duplicate asynchronously or synchronously tripolar or tetrapolar spindles will form.

"Whether the accumulation of chromosomally abnormal cells due to abnormalmultipolar spindle formation leads to embryonic arrest or whether embryonic arrest is the cause behind cytokinetic failure and multipolar spindle formation warrants further investigation," said Dr. Chatzemeteliou.

"Centrosomes are inherited via the sperm, so in future we hope to compare spindle abnormalities in embryos from patients with male factor infertility to embryos from patients with other fertility problems. We also hope to look at the cytoskeleton of embryos cultured in a number of different media so that we can investigate whether different culture conditions have an effect on spindle formation." Furthermore it would also be interesting to investigate, whether factors in the oocytes are responsible for spindle abnormalities and if cytoplasmic donation from good quality eggs, which is thought to improve embryo quality, could alleviate them.

Implications of this research for patients could be considerable. "Our work has considerable importance for patients undergoing preimplantation genetic screening for aneuploidy (an incorrect number of chromosomes), said Dr. Chatzimeletiou. "We have shown that the result obtained from a single biopsied cell might not be valid for the whole embryo due to mosaicism (the presence of two or more cell lines which differ from each other in chromosome number or structure, in an individual that has developed from a single fertilized egg).

For patients who are undergoing preimplantation genetic diagnosis (PGD) for a homozygous recessive gene disorder (where an abnormal disease causing gene is inherited from both parents), there could be a serious misdiagnosis of a carrier as being affected if the chromosome containing the normal gene is lost in the biopsied cell through a chromosome assembly defect." On the other hand, in a dominant disorder, where one parent has a disease-causing gene that dominates its normal counterpart, loss of the affected chromosome in the biopsied cell could result in the embryo being wrongly diagnosed as normal.

The identification of embryos with abnormal spindles using polarising microscopy, could provide a powerful non-invasive method for better embryo selection, which may lead to increased implantation rates. "Rather than simply rejecting embryos with spindle abnormalities", said Dr. Chatzimeletiou, "we are hopeful that in future it may be possible to rescue them by identifying those cells that contain the abnormal spindles and removing them from the embryo by biopsy. This could ensure a greater embryo conserve and higher chance for implantation. "