Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation

S. Stigliani, P. Anserini, P. L. Venturini, P. Scaruffi

Research output: Contribution to journalArticlepeer-review

Abstract

STUDY QUESTIONIs the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features?SUMMARY ANSWERThe mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤35 or >35 years old.WHAT IS KNOWN ALREADYCellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis.STUDY DESIGN, SIZE, DURATIONHuman fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study.PARTICIPANTS/MATERIALS, SETTING, METHODSDouble-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age.MAIN RESULTS AND THE ROLE OF CHANCEDifferent independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age.LIMITATIONS, REASONS FOR CAUTIONIt is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate.WIDER IMPLICATIONS OF THE FINDINGSThe approach we are proposing may provide a novel, non-invasive, objective tool for embryo quality grading. The correlation between a high mtDNA concentration and the fragmentation rate of embryos is suggestive that fragments are mainly anuclear cytoplasmatic debris arising during cleavage. Therefore, blastomere shaping as an early event during in vitro development may play a homeostatic role and be related to embryo competence.

Original languageEnglish
Pages (from-to)2652-2660
Number of pages9
JournalHuman Reproduction
Volume28
Issue number10
DOIs
Publication statusPublished - Oct 2013

Keywords

  • cell-free DNA
  • embryo fragmentation
  • human embryos
  • IVF
  • mitochondrial DNA

ASJC Scopus subject areas

  • Rehabilitation
  • Obstetrics and Gynaecology
  • Reproductive Medicine

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