ArrayCGH is a molecular technique based on the co-hybridization of sample and control genomic DNAs onto a microarray in order to identify gains and losses in sample DNAs (copy number changes). Generally it is used in post natal cytogenetics to identify small gains and losses. For more details click here.

What is copy number imbalance?

This refers to gains/losses of DNA in a sample. In IVF this is generally aneuploidy or gains/losses of whole chromosomes or large parts of chromosomes. For more details see here.

What is 24sure?

24sure is a single channel microarray system – not aCGH although similar - for analysing single cells or small numbers of cells for gains and losses of chromosomes – aneuploidy. Cells used in this system can come from various stages of embryo development; polar bodies (PB) from oocytes; blastomeres from day3 cleavage embryos; trophectodermal (TE) biopsy from blastocysts. The aim of the test is therefore to identify chromosomally normal or euploid embryos, with the rationale that euploid embryos are more likely to result in live births than aneuploid embryos, so improving IVF success rates.

Do BlueGnome have only BAC arrays within their product range?

Absolutely not. BlueGnome is a major supplier of oligo based microarrays for cytogenetic analysis of genetic disorders, also for oncology cytogenetics – see www.cytochip.com for more details. Recently we have also introduced the CytoChip Oligo SNP for combined genotyping and copy number applications. BlueGnome also produces has a full range of consumables and software developed in-house.

Why is 24sure better than FISH?

It has been clearly shown (multiple publications) that 24sure is more accurate and sensitive than FISH for PGD for aneuploidy. Also we have shown (internal data and data submitted for publication) that every chromosome is roughly as likely as any other chromosome to be aneuploid in an embryo. Hence FISH, even with the maximum 7-9 colours only detects a subset of aneuploidies from all aneuploidies seen and is therefore an incomplete test.

What is the clinical evidence that PGD aneuploidy testing improves pregnancy rates in IVF?

As yet the amount of the highest stringency clinical data – that obtained within randomized clinical trials – is limited. There are large case controlled studies and data from clinics to show that PGD for aneuploidy can greatly improve pregnancy rates for some indications of infertility, such as advanced maternal age and recurrent misscarriage. Although this work (see latest publications booklet) is highly encouraging it is not conclusive. Also work is being undertaken to determine which biopsy material is best for PGD aneuploidy testing. Since certain forms of biopsy are thought to be more detrimental to embryos than others, ie. where actual parts of the embryo (blastomere) that goes on to form the foetus are biopsied, compared to the less invasive biopsy (polar body and TE biopsy) where no material that goes on to form the foetus is removed. For instance when removing cells from the trophectoderm at day 5 and running 24sure, 3 separate groups have reported that if euploid embryos can be identified and re-implanted, then a higher pregnancy rate can be achieved, very largely independent of maternal age.

What is the most important factor for a PGD test for aneuploidy or monogenic disorders?

In order to run a PGD test for aneuploidy or monogenic disorders from a single cell the DNA must be amplified sufficiently to have enough DNA to analyse on an array. Thus requiring the approximately one million fold amplification of DNA from a single cell. This step is termed, whole genome amplification (WGA), and is the key step of PGD since if the amplified DNA does not faithfully represent the DNA of the starting cell then this can lead to false positive or false negative results. Regions of the starting cell that don’t amplify are called allele drop outs (ADO) and these can lead to false negatives. Similarly some regions of the starting cell may amplify better than others, leading to bias and possible false positives

Which whole genome amplification does BlueGnome use?

The WGA in 24sure is SurePlex. This has been developed by BlueGnome and Rubicon genomics. It is the only WGA specifically developed for single cell genomics and produces highly consistent and reliable results (amplification/reporting success of over 95%) with low ADO and consistent bias that can be controlled. Hence 24sure is a test with low false positive / false negative rates.

What types of samples (polar body, cleavage stage blastomere, blastocyst trophectoderm biopsy) are suitable for 24sure?

24sure can be used for any type of single human cell. From haploid (23 chromosomes) PB1/PB2, to diploid blastomeres and multi-cell trophectoderm biopsy. It can even be used to analyse single sperm. Using 24sure+ a higher probe density array, you can also look for imbalances in embryos of reciprocal translocation/robertsonian translocation carriers and inversion carriers.

Do you recommend just testing first polar body or testing of first and second polar body for aneuploidy PGD?

The data generated by BlueGnome suggests that since meiosis errors occur in both polar bodies almost equally, to stand the best chance of determining which embryos are euploid, both PB1/PB2 should be tested.

Why are BAC arrays better than SNP arrays for PGD of aneuploidy?

BAC arrays are better for some applications and SNP arrays better for other applications. One of the main advantages of 24sure’s BAC arrays is that they can be used with incredibly short hybridization times – down to 3hrs currently. This enables a highly time dependent process like PGD for aneuploidy to be run within a fresh IVF cycle. Even for the most time critical PGD for aneuploidy applications, such as polar body analysis (within certain central European countries) or day5 trophectodermal biopsy reported on the morning of day6, 24sure can be effectively employed. Even in instances such as countries where polar body testing is not limited to pronuclei formation, it is still advantageous to have a fast turn-around-time test since it allows the testing lab to follow embryo development until day2 and only test the polar bodies from appropriately developing embryos, thereby reducing the total number of polar bodies tested and costs. 24sure’s BACs have also been carefully optimised to give the lowest noise results with SurePlex WGA DNAs, hence results are clear, quick and easy to interpret.

How long does it take to run a 24sure array?

From sample biopsy to results 24sure can be performed in under 12hrs (for more information click here). Over 600 samples in a recent ESHRE clinical technology validation study were analysed by 24sure in under 12hrs.

Which PGD arrays are most sensitive to detect small chromosomal imbalances?

Results generated by 24sure+ have the smallest published change from a single blastomere cell. 24sure+ arrays can be tuned to detect small chromosomal imbalances and the smallest reported imbalance, so far, was less than 2Mb. It would be possible to design a 24sure+ array so that sub-megabase imbalances could be detected across the genome, however at this level of resolution you start to detect copy number imbalances found in normal population variation. These small imbalances are found in every normal human and hence having to interpret these copy number changes in embryos is highly undesirable.

What forms of imbalances can be detected using 24sure+ from embryos of Robertsonian/Reciprocal translocation carriers and carriers of other complex chromosomal abnormalities?

Sub 2Mb imbalances can be detected in unbalanced embryos of Reciprocal translocation carriers, however where no copy number change is present in the embryo, it is not possible to determine whether the embryo is balanced or euploid. Both balanced and euploid embryos are just as likely to result in a normal live birth.

Why do some test providers need parental DNA for PGD for aneuploidy?

WGA from single cells works at a lower efficiency for SNP arrays than BAC arrays. Hence most SNP arrays, when run for PGD have to use parental data to support results from single cells by filling in where data is absent due to allele drop out (ADO). This isn’t an ideal situation since effectively you are extrapolating from parental data to single cell data. Ideally you would run SNP array PGD without parental DNA however single cell WGA is not sufficiently representative to do this robustly. Karyomapping™ is different since it uses multiple runs of single cell SNP data, not single SNPs, to determine haplotype and the parental data to identify the ADO.

Can 24sure detect triploidy?

24sure can detect the most frequent triploidy XXY (~2/3 of all triploidy), but not the minor XXX triploidy. However, embryos are checked for pronuclei (PN) on day1, to ensure that diploid 2PN embryos are transferred not 1PN (haploid), 3PN (tripoid) or 4PN (tetraploid) embryos, therefore, this should not be an issue. SNP arrays cannot detect tetraploidy.

What lab equipment do I need to run 24sure?

To run 24sure requires a microarray scanner and some other general laboratory equipment. Expect this to cost from approximately $60-70K upwards depending on global location. Please contact BlueGnome and we can provide full list of equipment and costings. Also see here for more information.

Can you obtain an aneuploidy and monogenic disorder results at the same time from SurePlex?

Yes you can perform both PGD for aneuploidy screening and monogenic disorder testing on SurePlex WGA derived from a single cell. The PGD is performed in parallel using STR based marker testing – please contact BlueGnome for further details.

What types of arrays are suitable for monogenic disorder PGD?

In order to perform a monogenic disorder PGD test on an embryo you have to determine which genetic material (alleles) have been inherited from which parent so that an affected embryo is not transferred. In order to do this you have to genotype the embryo using a SNP (single nucleotide polymorphism) array. BlueGnome is developing Karyomapping™ which uses parental DNA and looks for runs of SNPs (haplotying) in embryos to determine which alleles are present. This technique has the promise to allow PGD to be run for many different disorders without the need to develop a new (STR) test for each case.

I don’t have the expertise to biopsy embryos for PB1/PB2, blastomeres or biopsy trophoectoderm?

BlueGnome can assist you with biopsy training. Please contact BlueGnome for further details.

Are there any published papers that use 24sure?

There are over 50 published papers and meeting abstracts on 24sure and Karyomapping. Please click here for a full list of the 24sure literature or download the latest publications booklet. BlueGnome customers can also login to see other customer presentations at www.cytochip.com/presentations.

How many staff and what qualifications are needed to run 24sure?

At a minimum two staff are required at different parts in the protocol to witness tube transfers (SurePlex to labelling reaction, labelling reaction to array). However one person can run the majority of a 24sure experiments. An experienced lab technician, proficient with setting up tests and able to perform tasks with accuracy and reliability, is preferred, although BlueGnome has trained 24sure users with limited previous lab experience.

How many samples can I run in a week?

This is dependent to some extent and dependent on how many staff there are in a lab, however one person can run up to ~30 samples per day. The precise number of samples is to some extent also dependent on how many samples there are per case, since each SurePlex WGA needs setting up separately for each case.

I have never run this kind of test before, will support and guidance be provided?

BlueGnome can provide user training courses either in our lab in Cambridge UK or globally at customer sites. Once customers start running cases we can also provide live support. Click here for more details.

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