Related Experiment Video
Updated: May 9, 2026
Human Blastocyst Biopsy and Vitrification
Published on: July 26, 2019
Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation
S Stigliani1, P Anserini, P L Venturini
1Center of Physiopathology of Human Reproduction, Unit of Obstetrics and Gynecology, IRCSS A.O.U. San Martino IST.
Study Question:
Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features?
Summary Answer:
The 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 Already:
Cellular 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, Duration:
Human 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, Methods:
Double-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 Chance:
Different 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 Caution:
It 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 Findings:
The 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.
Study Funding/Competing Interest:
This project was funded by Merck Serono (Grant for Fertility Innovation 2011). The sponsor had no role in study design, data collection, data analysis, data interpretation and writing of the paper. Authors declare no conflicts of interest.
Trial Registration Number:
ClinicalTrials.gov Identifier: NCT01397136.
Insights
Mitochondrial DNA (mtDNA) in embryo culture medium correlates with embryo fragmentation, offering a new objective marker for assessing embryo quality. This finding may improve in vitro fertilization success rates.
Area of Science:
- Reproductive biology and genetics
- Assisted reproductive technologies (ART)
- Embryology and developmental biology
Background:
- Cellular fragmentation is a key morphological indicator of human embryo quality and implantation potential.
- The precise origin and significance of cellular fragments in embryo culture remain unclear.
Purpose of the Study:
- To investigate the correlation between cell-free DNA (cfDNA) levels in human embryo culture medium and embryo morphological features.
- To determine if cfDNA can serve as an objective marker for embryo quality assessment.
Main Methods:
- Human fertilized oocytes were cultured individually from Day 1 to Days 2 and 3.
- Quantified double-stranded DNA (dsDNA), genomic DNA (gDNA), and mitochondrial DNA (mtDNA) in 800 spent embryo culture media using Pico Green assay and quantitative PCR.
- Statistical analyses correlated DNA content with embryo morphology (fragmentation) and maternal age.
Main Results:
- Both gDNA and mtDNA were detected in the embryo secretome.
- Higher DNA content was observed in embryos with poor cleavage quality compared to high-grade embryos.
- mtDNA concentration showed a significant association with embryo fragmentation rates (P = 0.0247) and advanced maternal age.
Conclusions:
- Embryo culture medium DNA profiles, particularly mtDNA, are significantly associated with embryo fragmentation, suggesting fragments are anuclear cytoplasmic debris.
- This approach offers a novel, non-invasive, objective tool for embryo quality grading.
- Further research is needed to validate its utility in predicting blastulation, implantation, and pregnancy rates.
