Session 27: PGTA
Questions for Darren
- What extra work up is needed using Karyomapping compared with the other techniques?
Questions for Andrea
- Lara Martínez: regarding mosaicism, how does the embryo’s ability of correcting its genetic mistakes come into account?
- martina cipriettiIs: it possible to make an analysis to detect abnormalities also from the blastocoele fluid?
- JC: Why did the STAR trial lack patients over 40 and emphasized patients under 35?
- Andrew Thomson: Given the wide variation in PGT-A success between clinics, do you think it could be bad technique/training that prevents PGT-A becoming more widely adopted/accepted?
- Bill Venier: Wouldn’t you expect some non-concordance to occur due to the fact of mosaicism and your getting more Representative DNA of the whole embryo?
- Andrea: That is an interesting point; using medium as a diagnostic tool could theoretically end up being a better predictor of the entire embryo (as opposed to simply a cohort of cells). Hower, this is a theoretical discussion at this point in time, and we still have a lot of work ahead of us.
- Prochi Madon: What is PGT-P?
- Andrea: It's a novel preimplantation genetic test for polygenic diseases/disorders. It's very exciting and the topic for the 28th i3 seminar!!
- The debate on mosaicism does not take into account that mosaic cells are probably disproportionately distributed between extra-embryonic tissue such as trophectoderm and most of the inner cell mass. Only a few cells in the mouse ICM are involved in fetal formation (probably just three cells). This means that the blastocyst is not a good organism to prove absence or presence of mosaicism.
- Andrea: While there is a vast body of literature demonstrating that mosaicism exists in human blastocysts, at this point in time there is little evidence supporting the notion that the aneuploid cell compartment is preferentially localized to the trophectoderm lineage. Again, more studies are needed to explore this question.
- Is cell free DNA perhaps “second class DNA” (denatured/damaged) and not truly representative of the Embryo Genome?
- Andrea: I think this is absolutely possible - the jury is still out. It's important for us to better characterize the mechanism by which this cfDNA is derived, for one thing; or possibly figuring out a way to sort the 'good/representative' DNA from the 'bad'. Still lots of work pending on this.
- Raifa Zody: Is there any harm in hatching on day 4 for TE biopsy?
- Andrea: I don't think there is much harm in hatching on D4, if performed correctly. I'm more concerned with the time the embryos are removed from their incubator to undergo the procedure.
- Sung Tae Kim: For non-invasive PGT from blastocyst, is artificial collapsing clitical to collect enough DNA or not?
- Andrea: For standard PGT-A and trophectoderm biopsy, artificial collapsing of the blastocoel cavity is not important/relevant (in fact it could confound the downstream data if you're not careful with washing your biopsy piece before loading it into a tube). Collapsing the blastocyst and including the blastocoel fluid as part of the non-invasive (or in this case, minimally-invasive) assay - so, for example, pooling this with the culture medium, has proven positive for some groups. These groups have been able to achieve high concordance between the blastocoel fluid plus culture medium and standard trophectoderm biopsy. But this is still being evaluated.
Questions for Joe Leigh
- Although viable spontaneous mutations during reproduction are rare, once all the mutations are added up, the total incidence is higher than some major anomalies like trisomy 21. The mutations can occur in gametes, early embryos or later embryos. Do you think that in the future De Novo mutations will be assessed routinely during IVF?
- Charles Muller: If the assumption that cfDNA from the fetus is from apoptotic cells, would that not increase false positive results?
- Joe: Cell free DNA due to apoptotic cells will result in a greater amount than expected based on reference amount once adjusted per fetal fraction. However, the next step is to determine the amount contributed by a given chromosome or chromosomes relative to the total DNA as well as to other chromosomes. Unless the fetal diagnosis is polyploidy, the chromosomes that stand out will represent true positives for aneuploidy.
- We know how important epigenetic processes are particularly important during early development. What about epigenetic profiling of embryos? Is that feasible and would it add to knowing/predicting the health of the embryo/fetus/child?
- Joe: Epigenetic profiling is an attractive explanation for many phenomena. The difficulty is our lack of knowledge concerning the regulatory phase of the genome, namely the 98.5% non-coding DNA . These sequences could be said to represent the “thermostat”, i.e., upregulating or down regulating the amount of gene product. The quantity of “regulation” that may have been appropriate in prior generations may no longer be appropriate as the environment ( food insecurity; pollution; climate ) changes. Methodology for quantifying this is not so well established analytically, nor are we close to a complete knowledge of the landscape.
- hui xu: what's the limitation of current pgt-M and what are desired new features for this test?
- Joe: Few limitations exist to PGT-M, providing we know the gene in question. Even if we do not know the actual mutation in a given case, linkage analysis can provide a diagnosis. The (DNA polymorphic) markers constituting the haplotype accompanying the mutant allele will differ from those accompanying the wild-type allele. I euploid miscarriage or in embryo selection, the shortcoming is that we know little about genes pivotal for differentiation. We can, however, cite genes that if perturbed result in zona pellucida abnormalities or zygote arrest as well as those (e.g., OCT 4 ) pivotal at the maternal to embryo expression transition. This is a burgeoning field in which knowledge will be expected to become available and, hence, testable for genetic perturbations