Mitochondrial DNA in day 3 culture medium as a molecular biomarker of human embryo fragmentation, aneuploidy, and implantation competency

Abstract Aim This study aims to use the mitochondrial DNA (mtDNA) in day 3 culture medium (D3CM) as a molecular biomarker of intracytoplasmic sperm injection (ICSI) and preimplantation genetic testing outcome. Methods This prospective observational study was performed using 49 embryos from 9 patients, which were individually cultured after ICSI. Day 3 culture media was collected, and blastocyst transfer or vitrification was carried out on day 5 of culture. Fold change of the mtDNA in D3CM (30 µl) was assessed using real-time PCR. The relationship between mtDNA fold change and different parameters of treatment outcomes was examined to find out if mtDNA in D3CM can be used as a predictive marker of embryo competency. Result The presence of mtDNA and genomic DNA was assessed in 49 D3CM samples, with only 1 sample found to be below the detection threshold. Among the 48 analyzable samples, mtDNA fold change demonstrated significantly greater predictive power for both aneuploidy and implantation outcomes than morphological assessment alone ( p = 0.001 and p = 0.001, respectively). Furthermore, mtDNA levels were significantly higher in D3CM samples from embryos exhibiting high fragmentation rates than those with lower fragmentation ( p = 0.004). In contrast, mtDNA content in spent culture medium was not significantly associated with blastocyst formation ( p = 0.336), blastocyst morphology evaluated by blastocoel expansion, inner cell mass quality, and trophectoderm appearance ( p = 0.787, p = 0.225, p = 0.433, respectively) or embryo sex ( p = 0.270). Conclusion The mtDNA content in day 0 to day 3 embryo culture medium may be a promising noninvasive tool for predicting embryo ploidy and competency. Higher mtDNA levels in D3CM and low/medium embryo fragmentation identified genetically normal embryos with high implantation potential. This suggests mtDNA profiling and fragmentation assessment can improve embryo selection in ICSI cycles.