Comprehensive chromosome screening and gene expression analysis from the same biopsy in human preimplantation embryos

Mol Hum Reprod. 2017 May 1;23(5):330-338. doi: 10.1093/molehr/gax014.


Study question: Can simultaneous comprehensive chromosome screening (CCS) and gene expression analysis be performed on the same biopsy of preimplantation human embryos?

Summary answer: For the first time, CCS and reliable gene expression analysis have been performed on the same human preimplantation embryo biopsy.

What is known already: A single trophectoderm (TE) biopsy is routinely used for many IVF programs offering CCS for selection of only chromosomally normal embryos for transfer. Although the gene expression profiling of human preimplantation embryos has been described, to date no protocol allows for simultaneous CCS and gene expression profiling from a single TE biopsy.

Study design, size and duration: This is a proof of concept and validation study structured in two phases. In Phase 1, cell lines were subjected to a novel protocol for combined CCS and gene expression analysis so as to validate the accuracy and reliability of the proposed protocol. In Phase 2, 20 donated human blastocysts were biopsied and processed with the proposed protocol in order to obtain an accurate CCS result and characterize their gene expression profiles using the same starting material.

Participants/materials, setting and method: A novel protocol coupling quantitative real-time PCR-based CCS and gene expression analysis using RT-PCR was designed for this study. Phase 1: six-cell aliquots of well-characterized fibroblast cell lines (GM00323, 46,XY and GM04435, 48,XY,+16,+21) were subjected to the proposed protocol. CCS results were compared with the known karyotypes for consistency, and gene expression levels were compared with levels of purified RNA from same cell lines for validation of reliable gene expression profiling. Phase 2: four biopsies were performed on 20 frozen human blastocysts previously diagnosed as trisomy 21 (10 embryos) and monosomy 21 (10 embryos) by CCS. All samples were processed with the proposed protocol and re-evaluated for concordance with the original CCS result. Their gene expression profiles were characterized and differential gene expression among embryos and early embryonic cell lineages was also evaluated.

Main results and the role of chance: CCS results from cell lines showed 100% consistency with their known karyotypes. ΔΔCt values of differential gene expression of four selected target genes from the cell lines GM4435 and GM0323 were comparable between six-cell aliquots and purified RNA (Collagen type I alpha-1 (COL1A1), P = 0.54; Fibroblast growth factor-5 (FGF5), P = 0.11; Laminin subunit beta-1 (LAMB1), P = 1.00 and Atlastin-1 (ATL1), P = 0.23). With respect to human blastocysts, 92% consistency was reported after comparing embryonic CCS results with previous diagnosis. A total of 30 genes from a human stem cell pluripotency panel were selected to evaluate gene expression in human embryos. Correlation coefficients of expression profiles from biopsies of the same embryo (r = 0.96 ± 0.03 (standard deviation), n = 45) were significantly higher than when biopsies from unrelated embryos were evaluated (r = 0.93 ± 0.03, n = 945) (P < 0.0001). Growth differentiation factor 3 (GDF3) was found to be significantly up-regulated in the inner cell mass (ICM), whereas Caudal type homebox protein-2 (CDX2), Laminin subunit alpha-1 (LAMA1) and DNA methyltransferase 3-beta (DNMT3B) showed down-regulation in ICM compared with TE. Trisomy 21 embryos showed significant up-regulation of markers of cell differentiation (Cadherin-5 (CDH5) and Laminin subunit gamma-1 (LAMC1)), whereas monosomy 21 blastocysts showed higher expression of genes reported to be expressed in undifferentiated cells (Gamma-Aminobutyric Acid Type-A Receptor Beta3 Subunit (GABRB3) and GDF3).

Large scale data: N/A.

Limitations, reasons for caution: Gene expression profiles of chromosomally normal embryos were not assessed due to restrictive access to euploid embryos for research. Nonetheless, the profile of blastocysts with single aneuploidies was characterized and compared. Only 30 target genes were analyzed for gene expression in this study. Increasing the number of target genes will provide a more comprehensive transcriptomic signature and reveal potential pathways paramount for embryonic competence and correct development.

Wider implications of the findings: This is the first time that CCS and gene expression analysis have been performed on the same human preimplantation embryo biopsy. Further optimization of this protocol with other CCS platforms and inclusion of more target genes will provide innumerable research and clinical applications, such as discovery of biomarkers for embryonic reproductive potential and characterization of the transcriptomic signatures of embryos, potentially allowing for further embryo selection prior to embryo transfer and therefore improving outcomes.

Study funding and competing interests: This study was funded by the Foundation for Embryonic Competence, Basking Ridge, NJ, USA. No conflicts of interests declared.

Keywords: ART; comprehensive chromosome screening; gene expression; preimplantation embryo; trophectoderm biopsy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy
  • Blastocyst
  • Cell Line
  • Chromosomes, Human / chemistry*
  • Chromosomes, Human, Pair 21 / genetics
  • Collagen Type I / genetics
  • Collagen Type I / metabolism
  • Down Syndrome / diagnosis
  • Down Syndrome / genetics
  • Down Syndrome / pathology
  • Embryo Culture Techniques
  • Embryo, Mammalian
  • Embryonic Development / genetics*
  • Female
  • Fibroblast Growth Factor 5 / genetics
  • Fibroblast Growth Factor 5 / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism
  • Gene Expression Profiling / methods*
  • Genetic Testing / methods*
  • Humans
  • Karyotyping
  • Laminin / genetics
  • Laminin / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Monosomy / diagnosis
  • Monosomy / genetics
  • Monosomy / pathology
  • Pregnancy
  • Preimplantation Diagnosis / methods*
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction


  • Collagen Type I
  • FGF5 protein, human
  • LAMB1 protein, human
  • Laminin
  • Membrane Proteins
  • collagen type I, alpha 1 chain
  • Fibroblast Growth Factor 5
  • ATL1 protein, human
  • GTP-Binding Proteins

Supplementary concepts

  • Chromosome 21 monosomy