Abstract
AbstractEarly embryogenesis involves a series of dynamic processes, many of which are currently not well described or understood. Aneuploidy and aneuploid mosaicism, a mixture of aneuploid and euploid cells within one embryo, in early embryonic development are principal causes of developmental failure.^1,2^ Here we show that human embryos demonstrate a significant rate of genetic correction of aneuploidy, or "genetic normalization" when cultured from the cleavage stage on day 3 (Cleavage) to the blastocyst stage on day 5 (Blastocyst) using routine in vitro fertilization (IVF) laboratory conditions. One hundred and twenty-six human Cleavage stage embryos were evaluated for clinically indicated preimplantation genetic screening (PGS). Sixty-four of these embryos were found to be aneuploid following Cleavage stage embryo biopsy and single nucleotide polymorphism (SNP) 23 chromosome molecular karyotype (microarray). Of these, 25 survived to the Blastocyst stage of development and repeat microarray evaluation was performed. The inner cell mass (ICM), containing cells destined to form the fetus, and the trophectoderm (TE), containing cells destined to form the placenta were evaluated. Sixteen of 25 embryos (64%) [95% CI: 44-80%] possessed diploid karyotypes in both the ICM and TE cell populations. An additional three Blastocyst stage embryos showed genetic correction of the TE but not the ICM and one Blastocyst stage embryo showed the reverse. Mosaicism (exceeding 5%), was not detected in any of the ICM and TE samples analyzed. Recognizing that genetic normalization may occur in developing human embryos has important implications for stem cell biology, preimplantation and developmental genetics, embryology, and reproductive medicine.1)Hassold, T. et al. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet. 44, 151-78 (1980).2)Menasha, J., Levy, B., Hirschhorn, K., & Kardon, N.B. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med. 7, 251-63 (2005).
Highlights
Text: Body Spontaneous miscarriages in human pregnancies have been well documented to be associated with chromosomal aneuploidy. 1, 2 In an attempt to minimize rates of aneuploidy in high risk pregnancies, single cells can be biopsied from early embryos and tested for their chromosome complement prior to uterine transfer. 3 This procedure, termed PGS, is generally performed on polar bodies or 1-2 totipotent blastomere cells biopsied from Cleavage stage embryos. 3 The traditional modality for evaluating the chromosomal makeup of these cells has been by fluorescence in situ hybridization (FISH) of 5-12 chromosomes.[3]
Data have failed to demonstrate that PGS by FISH on Cleavage stage embryos improve pregnancy outcomes and the delivery of healthy babies. 4,5 this approach has not become the recommended standard of care. 4,5 Potential reasons for the lack of demonstrated clinical benefit from karyotyping Cleavage stage embryos using FISH methodologies could be due to damage caused to the developing embryo during biopsy, testing of only a subset of chromosomes, or the presence of aneuploid mosaicism within the Cleavage stage embryo
Since 2007, comparative genomic hybridization (CGH) of metaphase chromosomes, real-time polymerase chain reaction (PCR), or microarray platforms using single nucleotide polymorphism (SNP) or CGH have been introduced to simultaneously evaluate all 23 pairs of chromosomes. 8,9,10,11 Use of 23 chromosome aneuploidy screening has been used to select embryos for uterine transfer with significant improvement in clinical pregnancy rates when compared to FISH methods that evaluate only between 5 and 12 chromosomes. 3, 11, 12, 13, 14
Summary
Summary IRB approval and patient consent was obtained for couples undergoing IVF with PGS. The Blastocyst stage ICM and TE microarray interpretations were performed blinded to all corresponding patient or embryo information These interpretations were repeated by the same blinded reader four separate times, confirming the integrity of the results with all data analyzed. A 23 chromosome molecular karyotype was obtained from over 99% of all blastomeres and all 31 cell lines The results from this validation data show that in this cohort of 110 abnormal embryos by Cleavage stage FISH, the vast majority contained some level of mosaicism. This may not be representative of studies conducted with much larger sampling size. Our lab has tested > 40 aneuploid mosaic cell populations and we detect monosomic and / or trisomic mosaicism at the level of 5%
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