Blastocoel fluid as a DNA source for PGD/PGS

April 17, 2013Carole No Comments »

In the most current issue of Reproductive Biomedicine Online, IVF researchers in Europe (S. Palini, L. Galluzzi, S. De Stefani, M. Bianchi, D. Wells, M. Magnani, C. Bulletti) have reported  in their paper “Genomic DNA in human blastocoele fluid”,(DOI: 10.1016/j.rbmo.2013.02.01) that:

1) they were able to recover embryo DNA from the fluid-filled center (the blastocoel) of blastocyst stage embryos and

2) they were able to amplify the DNA sample to determine the gender of the embryo and in some cases whether the embryo had abnormal chromosome numbers (aneuploidy).

This is exciting because it’s the first time that the blastocoel fluid has been found to contain at least fragments of DNA–which might be a good source of DNA for genetic analysis for preimplantation genetic screening (PGS) or preimplantation genetic diagnosis (PGD). PGS is a method for testing embryos for chromosome number abnormalities and for determining gender. PGD is a method for testing embryos for genetic sequence abnormalities (mutations) giving rise to genetic diseases or conditions. When abnormal embryo are determined, they can be preferentially excluded from transfer.

Currently, technicians perform embryo biopsy on each embryo to be tested. Technicians need to carefully remove one or more cells from the embryo to recover DNA for testing, which is tricky and if done poorly, can damage the embryo. In a day 3 eight cell embryo, taking 1-2 cells may result in slightly slowed progression as the embryo has very few cells to begin with and is at a delicate stage of development. It is technically simpler, quicker and perhaps more kind to the embryo to insert an ICSI needle into the middle of a blastocyst and remove some fluid, compared to carefully pulling off a small number of cells.

Basically, this needle stick into the blastocoel is a routine measure done currently to temporarily deflate or collapse the blastocyst before vtrification for optimal freezing results. It would  take relatively less skill and only a little training beyond that needed for ICSI for most technicians to become proficient at this technique. If it were this easy to get good DNA for genetic testing, that would definitely improve the technique for sampling. But is the quality of DNA what is required to get good reliable test results?

There are a lot of questions yet to be answered.  Jacques Cohen, master of micromanipulation techniques and his colleagues (Gedis Grudzinskas, Martin H. Johnson), do a good job of outlining the questions that still need to be answered before we all get too excited about this possible replacement for biopsy in this article-in-press editorial, “Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD?” This editorial can be accessed from the RBO site for free; the actual research findings paper -in the same issue -requires payment to read more than the abstract.

Here are the main challenges to be overcome as put forth by Jacques Cohen and colleagues,  (copied below):

  1. “Does the procedure sample DNA that is representative of the embryo? Control samples were obtained from the culture droplets, but it is known that human DNA is often present in embryo-free droplets of protein-supplemented culture medium. A follow-up trophectoderm biopsy was not performed, but this is an obvious second series of experiments to compare cell-free DNA results with those from biopsies.
  2.  Could the DNA have been released from abnormal or degenerate cells that are often excluded from the embryo? If so, the DNA may not always be representative of intact cells from the embryo.
  3. Is the procedure truly non-invasive? Whereas the procedure may not extract cells, damage may occur during the manipulation process possibly affecting the viability of the blastocyst.
  4. Can it be proven that the piercing of the trophectoderm and suction of fluid does not inadvertently release cellular material from the blastocyst, which is then aspirated and analyzed? The authors exclude this alternative explanation theoretically, and they may well be correct, yet the assumption needs to be proven.
  5. If DNA is dislodged into the blastocoel, can it also be released through the pores in the zona pellucida into the immediate environment?
  6. If affirmative, can the procedure be implemented by culturing blastocysts in minimal volumes to sample the products externally without micromanipulation? Can such sampling be done before blastocyst formation?”

In any case, it is an interesting initial finding and if further validated may refine and make safer current DNA sampling methods for PGD or PGS.


© 2013, Carole. All rights reserved.

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