Relative mtDNA copy number in embryo spent culture medium is not a reliable biomarker of human embryo aneuploidy

Abstract

Abstract: Mitochondrial DNA (mtDNA) from embryonic cells is released into the spent culture medium (SCM) during cellular processes, providing a potential biomarker of embryo health. Analysing mtDNA levels in SCM enables a non-invasive evaluation of embryo quality and potential developmental abnormalities. In this retrospective study, we aimed to investigate the relationship between relative mtDNA copy number in embryo SCM and key factors, including embryo fragmentation, morphological quality and chromosomal abnormalities. Fertilised embryos produced through intracytoplasmic sperm injection were cultured to the blastocyst stage in an incubator. Embryo fragmentation was assessed on day 3 using the Istanbul criteria, while morphological grading was evaluated on day 5 using the Gardner criteria. On day 5, trophectoderm (TE) biopsies were performed for preimplantation genetic testing for aneuploidy, followed by embryo cryopreservation and collection of embryo SCM. The mtDNA was quantified using quantitative PCR. Statistical analyses using the Mann-Whitney U and Kruskal-Wallis tests (significance at P < 0.05) showed that relative mtDNA copy number did not significantly differ among embryos with fragmentation levels <10%, 10-25% and >25% (P > 0.05). For blastocyst grading, which evaluates the inner cell mass (ICM) and TE, no significant difference was observed in relative mtDNA copy number between grades B and C for ICM (P = 0.190) and TE (P = 0.289). Furthermore, a trend towards higher relative mtDNA levels was observed in aneuploid than in euploid embryos, although the difference was not statistically significant. Thus, relative mtDNA copy number in SCM may not accurately reflect embryo characteristics, such as fragmentation, morphological grading or chromosomal abnormalities.

Lay summary: This study examined whether the amount of mitochondrial DNA (mtDNA) in the fluid used to culture embryos in the laboratory could indicate embryo quality. We assessed various factors, including the appearance of the embryos, the presence of fragmented cells and the occurrence of chromosomal abnormalities. Fertilized eggs were cultured until they developed into blastocysts, and the amount of mtDNA in the culture fluid was measured using a machine that detects genetic material. The results revealed no clear association between mtDNA levels and embryo appearance or fragmentation. Although embryos with chromosomal abnormalities had slightly more mtDNA, the difference was not statistically significant. These findings suggest that mtDNA in the culture fluid may not be a reliable marker for assessing embryo quality or chromosomal status.

Keywords: chromosome abnormality; embryo spent culture medium; fragmentation; mitochondrial DNA copy number.

Figure 1

Embryo morphology on day 3 and day 5. (A) The degree of embryo fragmentation was assessed using inverted microscopy and categorised into three groups. Scale bar = 100 μm. (B) Blastocyst grading was performed using inverted microscopy. The ICM is indicated by a star, while the TE is marked by a black arrow. Scale bar = 100 μm.

Figure 2

Box plots of relative mtDNA copy number and various embryo quality factors. (A) The relative mtDNA copy number in embryo SCM was significantly higher than that in the fresh medium (*P < 0.05). (B) The relative mtDNA copy number across the three fragmentation categories shows no significant differences. The relative mtDNA copy number with both the blastocyst ICM (C) and TE grades (D) shows no significant differences. (E) The relative mtDNA copy number with preimplantation genetic testing for aneuploidy revealed no significant differences.