Mitochondrial DNA content as a viability score in human euploid embryos: less is better  Antonio Diez-Juan, Ph.D., Carmen Rubio, Ph.D., Carlos Marin, B.Sc., Sebastian Martinez, B.Sc., Nasser Al-Asmar, M.Sc., Marcia Riboldi, Ph.D., Patricia Díaz-Gimeno, Ph.D., Diana Valbuena, M.D., Ph.D., Carlos Simón, M.D., Ph.D.  Fertility and Sterility  Volume 104, Issue 3, Pages 534-541.e1 (September 2015) DOI: 10.1016/j.fertnstert.2015.05.022 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Mitochondrial DNA (mtDNA) content quantifications. (A) The nuclear DNA (β-actin) and ATP8 regression curves used for assay normalization. The mtDNA and nuclear DNA (nDNA) copy number were calculated from the cycle threshold (Ct) number using the standard curve (1/Ct = 0.0077ln (nDNA) + 0.0159; 1/Ct = 0.0057ln (mtDNA) − 0.0139). (B) Histogram showing the sample Mitoscore (Ms) distribution in the four study groups. (C) Box plot corresponding to Ms versus implantation status. Wilcoxon rank-sum test was used to evaluate the difference in mtDNA content among the groups. ∗,#,&P<.02; @P=.02. Fertility and Sterility 2015 104, 534-541.e1DOI: (10.1016/j.fertnstert.2015.05.022) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Quartile sample distribution results for Mitoscore (Ms). (A) Ms in day 3: MsA (n = 51) implantation rate (IR) of 59%; MsB (n = 52) IR of 44%; MsC (n = 50) IR of 42%; and MsD (n = 52) IR of 25%. In embryos with Ms >160 (n = 22) all failed to implant. (B) Ms in trophectoderm: MsA (n = 16) IR of 81%; MsB (n = 16) IR of 50%; MsC (n = 16) IR of 62%; and MsD (n = 17) IR of 18%. With Ms >60 (n = 7) all failed to implant. (C) Pairwise t test differences, day-3 MsA and MsD (∗P<.02); MsA, MsB, and MsC versus MsD (@P<.02). Day-5 pairwise t test shows differences between MsA and MsD (#P<.02); and MsA, MsB, and MsC versus MsD (&P<.02). Fertility and Sterility 2015 104, 534-541.e1DOI: (10.1016/j.fertnstert.2015.05.022) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Mitochondrial DNA (mtDNA) content in dual male–female embryo transfer. Normalized mtDNA content is shown for two simultaneously transferred embryos (one male and one female) that resulted in a single pregnancy, along with the reported gender of the implanted embryo (n = 10). Each bar corresponds to an individual value from each embryo. The Mitoscore (Ms) is normalized and represented as a percentage of implanted embryo in each pair. (B) Box plot corresponding to Ms versus preimplantation genetic diagnosis indication category implantation status. AdA = advanced age; AF = abnormal FISH; IF = implantation failure; MF = male factor; RA = recurrent abortion. (C) Box plot corresponding to Ms in two age groups, ≤38 and >38 years. (D) Box plot corresponding to Ms versus embryo quality. Fertility and Sterility 2015 104, 534-541.e1DOI: (10.1016/j.fertnstert.2015.05.022) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Energetic stress induces changes in mitochondrial morphology and increases the Mitoscore (Ms). (A) JaR trophoblast cells were cultured in the absence or presence of a different concentration of the mitochondrial uncoupling compound 2,4-dinitrophenol (DNP): 75 (75 μM), 20 (20 μM), and C (control). Mitochondria (green) were detected by immunofluorescence with an antibody against complex V beta subunit (ATPB). Nucleus (blue) is stained with 6-diamino-2-phenylindole (DAPI). (B) Ms in JaR cells treated with DNP: 75 (75 μM), 20 (20 μM), and control. (C) A proposed model based on our findings. Top: In healthy embryos with a normal energetic reserve the mitochondrial biogenesis is low. The total amount of mtDNA is split between cells during division and thus the amount of mtDNA contained in individual blastocyst cells is lower than in cleavage-stage embryo cells; the overall embryonic mtDNA content remains the same. Bottom: In contrast, embryos with energetic stress activate mitochondrial biogenesis in an attempt to meet their energy demand; thus, the overall embryonic mtDNA content increases. Fertility and Sterility 2015 104, 534-541.e1DOI: (10.1016/j.fertnstert.2015.05.022) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions