Comprehensive Chromosome Screening (CCS)-panacea or pipe dream? - Part II

You can read the first part of this series here.

Karyotype showing aneuploidy of chromosome 21

 Embryo aneuploidy

An aneuploid embryo contains abnormal number of chromosomes in their cells. A human embryo should contain 23 pairs of chromosomes (46 chromosomes) in every cell. Chromosomes carry the genes. Genes encode the information for producing proteins. So when abnormal number (more or less)  of chromosomes are present in a cell more or less than the required amounts of proteins are produced. This can either kill the embryo by arresting its development (embryo doesn’t implant or even if it implants the pregnancy gets terminated spontaneously!) or can cause severe abnormalities in babies which are born out of an aneuploid embryo. For example a baby with Down’s syndrome carries three copies of 21^st chromosome (an entire extra chromosome or part of it!)  in its cells.

Aneuploid embryos can arise as the result of:

1) Errors in cell division during oocyte (egg) development.
2) Errors in cell division during sperm development.
3) Errors in cell division during an embryo development.

90% of embryo aneuploidy are due to cell division errors in oocytes and only 10% of the errors are contributed by the problems arising during sperm development. As a woman age the number of eggs with abnormal chromosome number increases.

Eggs and sperms are formed by a special type of cell division called meiosis.  The reason why we are so different from our parents and ancestors (in physical and mental traits) is because of genetic recombinations taking place during meiosis. Meiosis introduces genetic variations in offspring. Meiotic division is reponsible for the production of eggs and sperms. Eggs and sperms carry only half the number of chromosome (23 unpaired chromosomes) and during fertilization the full chromosome complement (46 chromosomes) is restored. When a chromosomally abnormal egg is fertilized by a normal sperm or vice versa (or if a chromosomally abnormal egg is fertilized by a chromosomally abnormal sperm) the resulting embryo will be genetically defective or aneuploid. When chromosomal errors occur during meiosis it affects the entire embryo. All the cells within the embryo will be aneuploid. Aneuploidy of meiotic origin is almost always lethal to the embryo or to the fetus. But approximately one third of aneuploid oocytes derived from sequential errors in the first and second meiotic divisions resulted in a balanced karyotype, representing a possible phenomenon of “aneuploidy rescue”

An embryo can also acquire genetic errors during its cell division. The embryo divides by a type of cell division called mitosis. The first three cell division of an embryo is extremely prone to genetic errors. An embryo activates its own genome when it reaches the 8-cell stage. Until then the embryo depends on maternally derived gene transcripts and proteins stored in the oocyte. For the prevention of aneuploidy formation high levels of mitotic and cell cycle proteins are necessary. The quality of the stored gene transcripts and protein could diminish over time by the accumulation of radiation or toxic agents, oxidative stress, compromised mitochondria or telomere shortening. This can lead to a defective cell cycle checkpoint mechanisms (especially in women of advanced maternal age), which may lead to chromosomal segregation errors in the first few cell division of human preimplantation embryos. When a zygote (embryo in 2pn stage) divides it gives rise to a two celled embryo. When genetic error occur in the first division then the entire embryo will be aneuploid. When a two celled embryo divides one cell division can be normal and the other defective. If such an error occurs one-half of the embryo will carry cells which will contain chromosomal abnormality and the other half of the cells will have a normal genetic make-up. When one-quarter of the cells in an embryo are chromosomally abnormal then it means that the chromosomal aggregation error occured in the third division. An embryo which carries both genetically normal (euploid cells) and geneticall abnormal cells (aneuploid cells) is said to be mosaic and the phenomenon which leads to the formation of mosaic embryos is called mosaicism.

Can mosaic embryos develop into normal babies?

Single cell comparative genomic hybridization analyses of normal IVF embryos showed that 75% of all the IVF embryos were mosaic. Out of these embryos 55% were diploid-aneuploid mosaic and 55% of all blastomeres from these embryos were diploid (PMID: 21531753). Mosaicism is found to be more in blastocysts when compared to cleavage stage embryos. This implies that mitotic errors could have occurred in later cell divisions and not necessarily in the first three mitotic division of a zygote. High rate of diploid-aneuploid mosaicism in blastocysts also implies that mosaic embryos more easily reach blastocyst stage as compared to embryos containing only aneuploid blastomeres. Do these mosaic embryos develop into normal babies? An experiment conducted in mice showed that only 20% of euploid cells in the blastocyst are enough to give rise to a normal mouse. This shows that blastocysts might have inherent mechanisms to correct their genetic defects. There are many speculations regarding such mechanisms. It is assumed that the blastocyst rearrange their blastomeres in such a way that the aneuploid cells are pushed towards the periphery forming the trophectoderm leaving only the diploid or euploid cells in the inner cell mass. This could explain the phenomenon of confined placental mosaicism. The other school of thought is abnormal cells are removed from the blastocysts via apoptosis and only the euploid cells survive. This leads to the accumulation of normal cells in the embryo.

It was also shown that frozen-thawed human embryos that lost nearly half of their blastomeres are still able to result in live births. This clearly shows that not all blastomeres of human pre-implantation embryos are necessary for developing into a full-fledged baby. Transfer of two tetraploid blastocysts (as identified by trophectoderm biopsy) in a woman has resulted in the birth of a normal male infant (PMID: 19608167). Embryonic stem cell lines developed from aneuploid embryos were found to have normal chromosomal make-up.These evidences clearly show that mosaic embryos have the potential to ‘self-correct’ and develop into normal babies. Might be, the type of mosaicism, the percentage of mosaic cells in an embryo and the inherent ability of the embryo to correct itself, determines whether a mosaic embryo could develop into a baby or not.

Further researches are needed at this point to determine whether embryo mosaicism is actually a pathological or physiological mechanism.

 

Array Comparative Genome Hybridisation

Fluorescence in situ hybridization (FISH) used to be technique for screening genetic abnormalities in pre-implantation embryos. However, this was unable to screen all the chromosomes in an embryo for genetic abnormality ; and was  also highly labor intensive. This has led to the development of advanced cytogenetic techniques which can scan all the 23 pair of chromosomes for genetic errors. One such technique is called array comparative genome hybridization (aCGH). The primary advantage of CGH is its ability to detect aneuploidies, deletions, duplications and/or amplifications of any locus represented in an array. One assay using this technique is equivalent to thousands of FISH experiments. Array-CGH has been successfully used to detect submicroscopic chromosomal aberrations which are also called as copy number variants (CNV).

Advantages of aCGH

1) Useful for comprehensive chromosome screening (CCS).

2) It has very high resolution (can detect submicroscopic variations in genome).

3) Quicker results and it is not labor intensive, since it is automated

Limitations of aCGH 
 
1) Whole genome screening using aCGH can generate data that may be difficult to interpret.

2) It can detect even minute alterations in genome which might have no established clinical relevance.

3) Clinical confidence of aCGH is still in question. Many researchers advocate FISH confirmation of the results obtained using aCGH.

You can read the next part here.