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Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD?

Open AccessPublished:March 18, 2013DOI:https://doi.org/10.1016/j.rbmo.2013.03.001
      The clinical usefulness of preimplantation genetic diagnosis (PGD) and screening (PGS) may be imperative, but it is not without its shortcomings. A number of arguments, some reasoned and others not, have been put forth against these technologies. To avoid transmission of genetic disease, patients and their doctors who may consider assisted reproduction, often choose post-conception testing with the option of termination of an affected pregnancy rather than commit to PGD. Moreover, while PGS may offer a method of selection for chromosomally normal embryos, alternative embryo selection modalities are usually favored over this technique. The invasiveness of many biopsy procedures is the most important reason why specialists consider other options first (
      • Cohen J.
      • Wells D.
      • Munné S.
      Removal of 2 cells from cleavage-stage embryos is likely to reduce the efficacy of chromosomal tests that are used to enhance implantation rates.
      ,
      • Kirkegaard K.
      • Hindkjaer J.J.
      • Ingerslev H.J.
      Human embryonic development after blastomere removal: a time-lapse analysis.
      ). Now there is a glimmer of hope that this situation could change.
      Researchers from Italy and England (
      • Palini S.
      • Galluzzi L.
      • De Stefani S.
      • Bianchi M.
      • Wells D.
      • Magnani M.
      • Bulletti C.
      Genomic DNA in human blastocoele fluid.
      ) have for the first time sampled blastocoelic fluid from expanded human blastocysts prior to vitrification and subjected the contents to PCR and DNA amplification procedures. The intervention did not involve cell biopsy. In an earlier study the content of the human blastocoel was analyzed by mass spectrometry in order to confirm the presence of molecular biomarkers produced by the embryo (
      • Poli M.
      • Ori A.
      • Turner K.
      • Child T.
      • Wells D.
      Defining the biochemical content of the human blastocoel using mass spectrometry: a novel tool for identifying biomarkers of embryo competence.
      ). The Palini team confirmed the presence of cell-free genomic DNA in the blastocoelic fluid of about 90% of the investigated embryos and several genes were identified related to the sex of the embryo. In a few instances chromosomes could be identified after whole genome amplification using a commercially available array CGH kit. Although there was an indication of the presence of fragmented DNA, genomic signals were sufficient to establish aneuploidy. The amount of DNA originally present in the sampled fluids was estimated to be equivalent to one embryonic cell. Although these findings are extraordinary, conclusions are still preliminary, since the quality of the DNA will need to be further investigated, as will the proportion of embryos in which a diagnosis will be possible.
      The advantages of performing PGD and PGS without biopsy are very obvious, but the procedure proposed by Palini and co-workers must be considered sensibly and potential clinical application must be approached with caution. A number of important questions should be addressed first by performing pre-clinical experiments using spare human blastocysts prior to clinical application: (i) 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. (ii) 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. (iii) 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. (iv) 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. (v) If DNA is dislodged into the blastocoel, can it also be released through the pores in the zona pellucida into the immediate environment? (vi) 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? The pursuit of this line of research will no doubt lead to answers of these and other relevant questions.
      The work of Palini and co-workers is provocative and fascinating for it raises the possibility of a new era in diagnosis of genetic abnormalities in preimplantation embryos.

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