Figure 7 miRNA and mRNA gene expression changes in the Poor Group

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Figure 7 miRNA and mRNA gene expression changes in the Poor Group Figure 7 miRNA and mRNA gene expression changes in the Poor Group. Transcript abundance for (A) sperm miRNA expression, and (B) blastocyst mRNA expression. Normalized fold change relative to the Good Group and to the internal constant MammU6 for miRNA expression, and to the housekeeping gene ACTB for mRNA expression. Statistical significance of P < 0.05 is denoted by an asterisk. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 1 Experimental outline and results summary Figure 1 Experimental outline and results summary. Normozoospermia sperm samples in donor oocyte IVF cycles resulted in either good quality (Good Group) or poor quality (Poor Group) embryogenesis. In the Poor Group, aberrant sperm DNA methylation in histone-retained regions was enriched for genes involved in critical developmental pathways and regulatory gene networks, while altered sperm miRNAs corresponded to altered target gene transcript levels in blastocysts, reflecting the poor blastocyst development observed. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 2 Sperm DNA methylation profiles of statistically significant genes for the histone-retained genome and across defined genomic regions. Mean methylation (β-values) for all significantly altered genes between the Good Group (black lines) and the Poor Group (red lines) distributed into 5% methylation intervals for (A) the histone-retained genome (1634 CpGs), (B–E) hypermethylated genomic regions: 5′UTR (189 CpGs), body (1185 CpGs), 3′UTR (130 CpGs), CpG shore (345 CpGs), (F,G) hypomethylated genomic regions: CpG island (311 CpGs), promoter (102 CpGs), (H) genes involved in embryo development and morphogenesis (81 CpGs) and (I) genes involved in transport and localization (290 CpGs). Statistics were performed for hypermethylated regions [80–100%], [80–90%], [90–100%] and hypomethylated regions [0–20%], [0–10%], [10–20%]. Statistical significance of P < 0.05 is denoted by *, and statistical significance of P < 0.001 is denoted by **. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 3 Regulatory gene networks identified for statistically significant genes. Top gene interaction networks generated from lists of genes with (A) increased methylation or (B) decreased methylation in the Poor Group. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 4 Representative sperm DNA methylation profiles at two imprinting control regions. A representation of targeted DNA methylation analysis in individual normozoospermic samples (n = 6) at the paternally expressed KCNQ1OT1 ICR; hypomethylated (Good Group: 1.2%; Poor Group: 1.0%), and the paternally methylated H19 ICR; hypermethylated (Good Group: 96.6%; Poor Group: 95.0%). No statistical difference was found between groups. Each line is a unique DNA strand amplified within the sperm sample. Black dots represent methylated CpGs, white dots represent unmethylated CpGs. Sperm ID is indicated below-left of the image. Percent methylation is indicated below-right of the image. Arrows indicate the CpG directly targeted by an Infinium HumanMethylation450 BeadChip probe, none of which were statistically significant between groups in the array dataset. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 5 Sperm DNA methylation validation for both groups at select genes. Targeted DNA methylation analysis in individual normozoospermic samples (n = 6) at 23 genes. Black diamonds represent sperm from the Good Group, and red diamonds represent sperm from the Poor Group. Each diamond is positioned at the average percent methylation for the individual sperm sample. Three methylation patterns are observed: hypermethylation in both groups, hypomethylation in both groups and variable methylation in both groups. From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Figure 6 Sperm DNA methylation percentages at ANKRD53 and MAPT Figure 6 Sperm DNA methylation percentages at ANKRD53 and MAPT. Targeted DNA methylation analysis in individual normozoospermic samples (n = 16) at ANKRD53 and MAPT. Black diamonds represent sperm from the Good Group, and red diamonds represent sperm from the Poor Group. (A) Each diamond is positioned at the average percent methylation for the individual sperm sample. (B) Average percent methylation is distributed by the 21 CpG positions at ANKRD53 for both groups (Good Group: 91.6%; Poor Group: 82.0%; P < 0.05). (C) Average percent methylation is distributed by the 19 CpG positions at MAPT for both groups (Good Group: 73.1%; Poor Group: 53.8%; P < 0.05). From: Alterations in the sperm histone-retained epigenome are associated with unexplained male factor infertility and poor blastocyst development in donor oocyte IVF cycles Hum Reprod. 2017;32(12):2443-2455. doi:10.1093/humrep/dex317 Hum Reprod | © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com