All cells in a human organism other than the germ cells (egg and sperm) contain 46 chromosomes which are arranged in pairs. One pair will be inherited from the mother’s egg (22, X) and the other from the father’s spermatozoon (22, X or Y). Chromosomes which are thread-like structures distributed within the nucleus of each cell contain the 20000-25000 genes which are the basic physical and functional molecules of heredity.Alterations in the number and structure of chromosomes associated with additions or deletions of genetic material in cells subject humans to birth defect risks and/or mental conditions which limit individual behavioural and intellectual capacity.
Pre-implantation genetic diagnosis is offered to couples who are at risk of transmitting an inherited disorder to their offspring. The type of genetic predisposition may be associated with a single gene defect (monogenic disorder) or a structural abnormality in one or more chromosomes. Individuals with chromosomal or single gene disorders are diagnosed prior to a potential treatment becoming in effect, either because of a known family history or due to recurrent miscarriages or following conceiving an affected child.
Couples who present such genetic predispositions may opt to benefit from the advents in PGD technology or choose from a number of alternatives such as egg or sperm donation, accept the accounted risk which may lead into a miscarriage or potential elective termination (following prenatal investigation) or transmitting the condition to their offspring, remain childless or adopt.
PGD in early developing embryos is not limited to the investigation of previously diagnosed genetic conditions. Rather, couples who wish to minimise the risk of achieving a defective conception may choose to receive chromosomal screening on their embryos prior to any of them being replaced into the uterus using a process adjunct to IVF/PGD namely PGS. Hence while PGD is associated with the screening of a known chromosomal or gene disorder, PGS can be implemented merely to enhance the chances of a healthy pregnancy resulting. Good candidates for PGS are couples with advanced maternal age, repeated failures to conceive using IVF and those who had suffered repeated miscarriages but with normal Karyotype patterns for both spouses.
Prerequisite to implementing PGD/PGS is the undertaking of IVF. As previously described this entails the ovarian stimulation phase and the fertilisation of the egg harvest. Prior to the embryo transfer, embryos are treated with biopsies for the removal of one or more cells. The embryo biopsy entails the creation of a small aperture in the outer shell of the embryo (Zona Pelucida) in order to facilitate the aspiration of the cell(s). While this was conventionally implemented with the aid of an acid solution (Tyrode), laser puncture has prevailed as the most effective and safer means.
Biopsies and subsequent PGD is not limited to embryos only. Rather, eggs can receive biopsies either before or following their fertilisation. Specifically, during its final maturation phase in the ovary, the egg’s 46 chromosomes are separated via a process called meiosis. At the time the immature egg transforms into two significantly different in size cells each containing 23 chromosomes. A small cell namely the polar body and the so called secondary oocyte. Following fertilisation the secondary oocyte will resume meiosis and this gives rise to a second polar body. Since neither of the two polar bodies contribute to the genetic complement of the embryo and eventually disintegrate, can be biopsied and therefore their chromosomal content or potential gene derangements investigated.
Embryo biopsies are conventionally performed on day 3 of in vitro development (6-8 cell stage) or on day 5 when the embryo is expected to have reached the blastocyst stage (several dozen cells). The blastocyst is structurally formed by an inner cell mass which subsequently integrates into the new organism. The outer layer of the blastocyst consists of cells collectively called the trophectoderm programmed to form the placenta. This layer surrounds the inner cell mass forming a fluid-filled cavity namely the blastocoele. By contrast to day 3 biopsies where only one or two cells are removed from the embryo the number is considerably higher when blastocyst biopsies are implemented.
The value of day 3 biopsies has received criticism pending the large volume of cellular material which is removed from the embryo (1/8 or 12.5%). Furthermore the diagnosis derived from a single cell may be false positive or negative due to the risk of chromosomal mosaicism, a phenomenon which increases with age.
Mosaicism in essence means that embryos are made up of more than one type of cell line. Healthy embryos can only develop from one line of cells while secondary cells (mosaic) are either incorporated into the trophectoderm or commit apoptosis (programmed cell death). Embryo viability is not compromised by an abnormal cell line being present in the trophectoderm. There is literally no determining criterion as to which of the 8 cells will be removed from a day-3 embryo. The selection is a measure of randomness. In this respect and by definition of mosaicism the chromosomal content of a single cell may be found to be normal while the majority of the embryo cells could have abnormal chromosomal complement(s) and vice versa. This is a common cause of misdiagnosis when using PGD/PGS.
Blastocyst biopsies mitigate this risk as sufficient number of cells (8-12) can be removed for an affirmative diagnosis to be made. When implementing biopsies on blastocysts the inner cell mass must not be manipulated during the procedure since this will seriously compromise embryo viability. Rather, the cells are biopsied from the trophectoderm area of the embryo where recovery can essentially be achieved. Albeit, as described above, since embryos segregate their abnormal cell lines into the trophectoderm, biopsying chromosomally deranged cells is a possibility. However, the large number of cells which become available for assessment following trophectoderm biopsy mitigates potential misdiagnosis
Hatched Blastocyst Biopsy
Fluorescent in Situ Hybridisation
DNA, the fundamental molecule in our cells which curries all the genetic instructions of functioning and reproduction while is structured amid two strands coiled together forming a double helix. This is held together by the binding interaction of four bases namely A, T, G and C. Each strand is complementary to the other where these bases can only bind together as A-T and G-C (and vice versa). FISH makes use of the specific binding ability (hybridisation) of one strand to connect to its counterpart.
Following biopsy the embryonic cell(s) is/are spread on special colloidal glass slides within an engraved region of interest (this assists at locating the cell nucleus during diagnosis). The spread on the colloidal-treated glass slides is managed with the use of an acid solution. Following washing the slides are incubated at a concentration of an enzyme (Pepsin) solution which aims at removing cytoplasmic debris exposing thereby the nucleus of the cell.
Fluorescently labeled synthetic DNA probes which are complementary to particular DNA sequences for the chromosomes to be screened are applied within the region of interest on the glass slide. Subsequent heating of the glass slide for 3-4 minutes at 73°C causes the nucleus’ DNA to open apart or denature allowing the fluorescently tagged probes to hybridise to their complementary sequence in the nucleus’ chromosomal DNA. This hybridization step is undertaken in a moist chamber at 37°C for a period which can range between 45 minutes to several hours.
Using a high magnification fluorescent microscope equipped with appropriate colored filters (corresponding to the synthetic DNA tags) the appearance of 2 distinct signals confirms a normal chromosomal complement (a male embryo is defined as one type of colored signal for the X chromosome and another for the Y). Additional or fewer fluorescent signals indicate a derangement for a particular chromosome.
Similarly, embryos which are screened for a segmental chromosomal aberration such as a reciprocal or a Robertsonian translocation and other inherited chromosome abnormalities a duplicate signal for the chromosomal region of interest indicates a normal or balanced chromosomal condition for the embryo whereas the presence of one or more than two signals is consistent with a chromosomally deranged cell. When screening for segmental chromosome abnormalities the use of three different fluorescent probes is recommended and this is because the aberration involves the rearrangement of parts between two non homologous (one from the mother and one from the mother) chromosomes.
In other words a two way exchange of information exists between the two chromosomes. Two of the fluorescent signals will correspond to the chromosomes associated with the condition while a third set will associate with the particular site of the mutation. The commonest reciprocal translocation is t(11;22). This designates that a part of chromosome 11 dislocates from its position and adheres onto chromosome 22. In this respect two fluorescent probes will be required, one for each 11 and 22 chromosomes and another which has been designed specifically to bind at the cut off site of chromosome 11.
Comparative Genome Hybridization (CGH) and Microarrays
CGH is a cytogenetic technique which endeavours to examine variations in the structure of chromosomes of a single cell or a cluster of similar cells such as embryonic cells. In essence it compares the number of sets of chromosomes of a given test specimen i.e. that of the biopsied cell(s) with that of a closely related normal reference sample based on the assumption that they contain differences in the context of gains or losses of either whole chromosomes (a condition namely aneuploidy) or subchromosomal regions (a part of a whole chromosome namely segmented chromosomal derangement).
As the resolution of traditional CGH has been limited to large genome alterations (approximately 5-10 Mb for most clinical applications) a newer method combining the principles of the technique with microarrays has been introduced to overcome this. This method namely aCGH uses microscope glass slides which are arrayed with very small DNA segments namely probes representing the targets of analysis. Such slides can be customarily prepared on the basis of a known predisposition associated with small regions of interest or to represent larger genomic clones therefore enabling the assessment of large chromosomal regions.
The method which encompasses either of the two approaches is literally the same. The first step in the process involves the extraction of DNA from the test sample i.e. embryonic or fetal cells. This is then labelled using a fluorescent dye of a particular colour which is different from the one used to label the control (reference) sample i.e. red and green. In the third step the coloured test and reference genomic DNAs are mixed together and applied onto the microarray slides. Pending the fact that these are denatured i.e. are in single strands, they will hybridize with the counterpart probes on the slides. Amid advanced laser imaging systems the relative intensities of the labelled DNA probes can be captured and quantified following hybridization and therefore the fluorescence ratio between the two can be determined at different positions along the genome. Therefore, information can be obtained regarding the copy number of sequences in the test genome as compared to the normal one. If a higher intensity is observed for the test sample colour for a specific region then this is consistent with a gain of a chromosome for that region while a higher intensity for the reference sample indicates the loss of a chromosome for the test sample for that region.
Similarly a neutral colour is a denominator of normality for the region of interest. Since aCGH detects an alteration in chromosome copy numbers it can only be useful for determining deletions, additions or duplications of copy numbers i.e. unbalanced chromosomal aberrations but not inversions or reciprocal translocation where the copy number remains unaltered.
Next Generation Sequencing (NGS)
Next Generation Sequencing (NGS) is a new, cutting-edge technology which can analyse small quantities of DNA harnessed from cells biopsied from early developing embryos and generate the full 23 pairs of chromosomes of a sample with remarkable resolution and sensitivity.
Initially, the sample undergoes Whole Genome Amplification (WGA), a process that essentially turns the original quantity of a sample DNA into a significantly higher amount which is suitable for testing.
The DNA sequencer can run 24 samples at the same time. The samples are processed in a rigorous, hands-on protocol through which the DNA is added on the sequencer for processing. As new DNA is synthetized, fluorescent signals are emitted. The software uses these signals to identify the specific DNA sequence formed and its location. Finally, all sequences created are separated based on the specific markers added on each. During data analysis, results are imported on a specialized software which generates the chromosomal profile of each sample.
As NGS is highly sensitive, it allows the detection of whole copy numbers as well as small segmental changes (deletions/duplications) to be clearly and accurately seen, a significant advantage over FISH or aCGH technologies. Additionally, NGS as a method of analysis is superior in the context of its capacity to detect mosaicism and its level in an embryo. Due to the sequencer having a minimum capacity of running 21 samples at a time, and the required time it takes to run, process and read the results, a Fresh Embryo Transfer is not currently possible. The embryos are frozen after biopsy for implantation at a later stage. The implantation potential of embryos which have been found healthy for all chromosomes after NGS testing is as high as 80% following successful thawing. This rate further increases with a confirmation of a foetal heart pulse or ultrasound at 6 ½ weeks.
The only disadvantage of NGS technology is its inability to diagnose ploidy status in female embryos, a rare phenomenon which is minimized even more by good IVF practices.