Genetic normalization of differentiating aneuploid cleavage stage embryos. P. R. Brezina1, R. Ross2, A. T. Benner3, R. P. Dicky4, R. Kaufmann2, R. Anchan5, Y. Zhao6, A. Barker7, K. J. Tobler6, G. R. Cutting8, W. G. Kearns3,6 1) Fertility Associates of Memphis, Memphis, TN; 2) Fort Worth Fertility, Fort Worth, TX; 3) Center for Preimplantation Genetics, LabCorp, Rockville, MD; 4) The Fertility Institute of New Orleans, Mandeville, LA; 5) Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, Brigham and Women's Hospital, Harvard Medical School, Boston, MA; 6) Department of Gynecology and Obstetrics, Division of Reproductive Endocrinology and Infertility, Johns Hopkins Medical Institutions, Baltimore, MD; 7) Arizona Center for Fertility Studies, Scottsdale, AZ; 8) McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins Medical Institutions, Baltimore, MD.
We determined if aneuploid embryos can undergo genetic normalization during differentiation into blastocysts. Forty patients underwent 40 in vitro fertilization cycles and preimplantation genetic screening secondary to repeat pregnancy loss or unexplained infertility. IRB approval was obtained. Single blastomeres from 325 cleavage stage embryos were biopsied and underwent DNA extraction and amplification by a modified multiple displacement amplification protocol and single nucleotide polymorphism (SNP) microarrays or a random priming DNA amplification protocol and comparative genomic hybridization (aCGH) arrays. All embryos were cultured to Day-5 or Day-6 following oocyte fertilization. If aneuploid embryos differentiated into a blastocyst, they underwent surgery to separate the inner cell mass (ICM) from the trophectoderm (TE). The ICM and TE cells were then confirmed by immunocytochemistry using Oct3/4 and Cdx2 respectively. Following cell type confirmation SNP or aCGH microarrays were performed. Forty-eight percent (156/325) of cleavage stage embryos were euploid. Of these, 70% (109/156) differentiated into blastocysts. Of these patients, 60% (24/40) achieved a clinical pregnancy with fetal cardiac activity. In contrast, 52% (169/325) of cleavage stage embryos were aneuploid and only 39% (66/169) differentiated into a blastocyst. Of these 66 blastocysts, 36% (24/66) remained aneuploid in both the ICM and TE cells, 5% (3/66) of the blastocysts had a euploid ICM and an aneuploid TE and 2% (1/66) of aneuploid blastocysts had a euploid TE but an aneuploid ICM. Remarkably, 58% (38/66) of the cleavage stage embryos with an aneuploid blastomere, that differentiated to the blastocyst stage of development, were euploid for all ICM and TE cells analyzed. No uniparental disomy was observed. These results demonstrate that a substantial fraction of cleavage stage embryos, with identified aneuploidy in a single blastomere, can normalize during differentiation to the blastocyst stage of development. We hypothesize that the aneuploid blastomeres from cleavage stage embryos are marginalized during differentiation. We propose that mosaic aneuploidy in early embryo development and subsequent embryo normalization to form euploid embryos is not an uncommon event in human embryo differentiation.
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