Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified  Elizabeth R. Hammond, B.Sc., Brent C. McGillivray, M.Sc., Sophie M. Wicker, M.Sc., John C. Peek, Ph.D., Andrew N. Shelling, Ph.D., Peter Stone, M.B., Ch.B., Larry W. Chamley, Ph.D., Lynsey M. Cree, Ph.D.  Fertility and Sterility  Volume 107, Issue 1, Pages 220-228.e5 (January 2017) DOI: 10.1016/j.fertnstert.2016.10.015 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The absolute number of mitochondrial DNA (mtDNA) copies in spent embryo media (n = 154) and media controls (n = 54) in day 3 and day 5 sequential G5 series medium (Vitrolife) and day 6 continuous single-culture medium. The spent sequential media from 51 embryos was sampled at day 3 of culture and then again at day 5 of culture and the spent continuous media from 52 embryos was sampled at day 6 of culture. The baseline level of mtDNA in media controls was 15 ± 4 copies, and there was no difference between the three types of culture media (P=.470). Compared with the media controls, there was a higher number of mtDNA copies in spent embryo culture media (P<.0001). The mean number of mtDNA copies in spent embryo media was 90 ± 22 at day 3 and 259 ± 42 at day 5 of sequential culture (P<.0001). The mean number was 625 ± 118 copies at day 6 in continuous culture (P<.0001). ****P≤.0001 (Wilcoxon matched-pairs signed-rank test for paired data, Mann-Whitney test for media control data and Kruskal-Wallis tests with Dunn's multiple comparison tests for comparison with continuous single-culture media). Fertility and Sterility 2017 107, 220-228.e5DOI: (10.1016/j.fertnstert.2016.10.015) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Cycle threshold (CT values) of mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), TBC1 domain family, member 3 (TBC1D3), testis-specific protein, Y-linked 1 (TSPY1), ribonuclease P (RNAse P), and cystic fibrosis transmembrane conductance regulator (CFTR) in media controls (n = 25) and spent embryo media (n = 100). The y axis is labeled in reverse to reflect how the CT level is inversely proportional to the logarithm of the initial level of DNA in the sample. Of those media samples that amplified during qPCR, the spent embryo culture medium had a higher abundance of MT-ND1, TBC1D3 (P<.0001) and CFTR (P=.019) compared with the media controls, but this was not significantly different for TSPY1 (P=.384) or RNAse P (P=.102). *P≤.05, ****P≤.0001 (Mann-Whitney test). Fertility and Sterility 2017 107, 220-228.e5DOI: (10.1016/j.fertnstert.2016.10.015) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Y/17 chromosome ratio in male controls (n = 20), media controls (n = 18), and spent embryo media (n = 81). Compared with the male controls, there was a higher Y/17 ratio in media controls (P=.029) and spent embryo media (P<.0001). Compared with media controls, spent embryo medium had the highest ratio, indicating an excess of female-derived DNA (P=.0005). *P≤.05, ***P≤.001, ****P≤.0001 (Kruskal-Wallis tests with Dunn's multiple comparison tests). Fertility and Sterility 2017 107, 220-228.e5DOI: (10.1016/j.fertnstert.2016.10.015) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Representative gel electrophoresis image of intact mitochondrial DNA (mtDNA). Amplification of the 15-kb band during long-range polymerase chain reaction indicates the presence of intact mtDNA in spent embryo culture medium. This representative image shows spent embryo culture media from two patients (sample 1 and sample 2) and respective media control droplets. The 15-kb fragment was not detected in spent embryo media from samples 1C, 2B, 2G, 2I, and 2J and showed no evidence of amplification in media control droplets. Fertility and Sterility 2017 107, 220-228.e5DOI: (10.1016/j.fertnstert.2016.10.015) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions