As discussed in early blogs, PGS and PGD are the techniques, which offer testing of the embryos before their transfer into the uterus. While PGS identifies the embryos with numerical chromosomal aberrations, PGD is a directed molecular diagnostic test for a particular gene of interest. In couples using those techniques, accurate and reliable determination of single gene defects, chromosome structure, and chromosome number is used to advise embryo selection before the embryo transfer. For many, this is much more desirable option than waiting for fetal diagnosis within the first or second trimester of pregnancy.
Towards the above techniques, the modern ways of molecular genetics began to appear too. Those are launched in Ukraine in some way.
Array Comparative Genomic Hybridization (CGH)
Array comparative genomic hybridization (CGH) is the technique that enables simultaneous and complete enumeration of chromosomes from a single biopsied cell without cellular fixation. With CGH, labeled DNA from both test and normal DNA samples are hybridized to a DNA microarray, which includes roughly 4,000 markers spaced throughout the entire genome. CGH does not directly visualize chromosomes but determines the relative copy quantity of chromosome between the test DNA along with a control normal DNA after concurrent PCR amplification.
The technique is capable of screening biopsied cells for chromosome copy number (aneuploidy) and unbalanced chromosome translocations. CGH may also be used for proper diagnosis of some translocations, inversions, along with other chromosome abnormalities where there is an increase or lack of chromatin. With respect to the size of probes used (bacterial artificial chromosome or oligonucleotide) some clinically important microdeletion or microduplication disorders can also be detected.
It will not differentiate balanced translocations unless there are subtle variations in DNA copy numbers that occasionally exist in them along with other chromosomal structural rearrangements e.g. inversions. CGH will also not differentiate whole genome ploidy states e.g. polyploidy or monoploidy as there is an equal representation of chromosomes. It is also not able to identify uniparental disomy. This technique has limited capability to identify mosaicisms only when the platform has been formerly validated against a mosaic cell sample. CGH platforms can amplify DNA and finish the analysis usually in 12-15 hours, thus lending itself to fresh or frozen embryo transfers.
SNPs are highly conserved variations in a single site within the DNA, which exist inside a frequency more than 1% inside a population. There are more than 40 million SNPs within the human genome, which have been validated, making it a very sensitive and specific genotyping marker for diagnosis. The microarray includes of a chip that contains nucleotide acid sequences complementary to every SNP region of interest (density coverage may vary from 600,000 to 2 million SNPs with respect to the chip).
The sample DNA acquired from PGD is amplified and hybridized to the chip. The hybridization signal detected can concurrently showcase DNA copy number essential for aneuploidy screening and recognition of clinically significant microdeletion and microduplications in addition to determine parental origin, existence of uniparental disomy, and lack of heterozygosity.
It is an effective molecular tool however with some limitations, particularly the lack of ability to recognize balanced translocations, or whole genome polyploidy. There might be many de-novo structural chromosomal abnormalities, which are underneath the resolution of the SNP array too. SNP arrays take usually 30-40 hours to complete the analysis, thus lending itself only to frozen embryo transfers.
Next-Generation Sequencing (NGS)
Next-generation sequencing (NGS) is a technology that utilizes enhanced, high-throughput DNA amplification to sequence DNA. The procedure involves fragmenting DNA into countless small fragments, which are then fused with an adaptor and a barcode to create a DNA library. The library will be loaded right into a flow cell where the fragments bind to a surface of complementary surface-bound oligonucleotides and then amplified to produce distinct clonal clusters. High-throughput, paired-end reversible terminator-based sequencing of the fragments detects single bases as they are integrated into the DNA template strands, reducing sequencing errors. Paired-end sequencing produces twice the number of reads that occur and the paired sequences are aligned as read pairs, further reducing the probability of errors in sequencing. The amplified fragments are aligned to a reference genome to identify variations between the fragment and the reference.
The attachment of the barcode allows for multiple libraries to be run simultaneously and then sorted before final analysis. Advances in NGS have reduced time for library preparation and time for sequencing. The ability to multiplex enables for scalable instrumentation with respect to the anticipated utilization. NGS will identify whole chromosome aneuploidy, mosaicism, triploidy, large deletions, or duplications more than 50 Mb, some clinically significant deletions or duplications 800 b to 1 Mb, uniparental disomy, and mitochondrial copy number.
In conclusion, CGH, SNP Microarray and NGS are the rapidly expanding modern technological advances that could greatly benefit couples of risk of transmitting genetic or chromosomal abnormalities for their offspring. As much evidence become available in Ukraine, as much popular the use of modern methods of molecular genetics will be, proving to be beneficial to many couples who use assisted reproductive technologies (ART).