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Introduction MtDNA copy number analysis remains controversial as a biomarker for embryo implantation and viability assessments with different authors reporting conflicting results. Initial reports ( Fragouli et al., 2015 , Diez-Juan et al., 2015 ) indicated that embryos with unusually high levels of mtDNA did not implant and those that did have a mean lower mtDNA count. Such findings have been challenged in different cohorts however ( Treff et al., 2017 , Victor et al., 2017 ). The purpose of this study was to determine if an association exists between mtDNA content and 3 clinical parameters: female age, the day of biopsy and implantation outcomes in euploid embryos in a large, blinded, cohort. Materials and Methods Aneuploidy testing was performed on 3971 blastocysts obtained from 1717 IVF/ICSI cycles in a single IVF unit, for patients with repeated implantation failures, recurrent miscarriages and/or advanced maternal age. Next generation sequencing (NGS) technology (Thermo Fisher) assessed chromosomal status. Euploid embryos were further analyzed by Ion Reporter software to identify mtDNA content by using the algorithm Mitoscore©. Blastocyst biopsy was performed by removing 4-8 trophectoderm cells, then blastocycsts were vitrified and transferred in a programmed thaw-cycle in the presence of euploid embryos. Embryos were divided by maternal age into “40 and over” and the remainder. Association between mtDNA and implantation was assessed in cycles where only one embryo was euploid so not to be affected by variables such as Mitoscore and/or morphology. Statistical analysis was performed by t-test (p Results 1866/3849 (48%) of embryos were euploid, with 681 cycles (39.6%) cancelled as none were diagnosed as such. Of 1838 where Mitoscores© were available, euploid embryos in the ≥40 age group (n=286) were more likely to have increased amounts of mtDNA copy number compared to younger group (n=1552)(P Conclusions In a previous preliminary study, we found no significant difference in mtDNA content between implanted vs. not-implanted embryos, however this may have been due to small sample size. After doubling patient number we now report a statistically significant difference (p=0.03) suggesting some value to Mitoscore in our hands. Nonetheless, outliers still exist as embryos with high Mitoscores still implanting, bringing into some doubt claims of a negative predictive value. This study has advantages over previous ones in that it is a large, blinded study that was performed in a single setting where results are less likely to have been confounded by variables such as stimulation protocols and laboratory conditions. Discrepant results may arise due to variability in data sets, different assessment protocols, software and algorithms. Further research on larger sample sizes in a prospective blind setting are needed in future.