Agnieszka Malcher, M. S. , Natalia Rozwadowska, Ph. D

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Potential biomarkers of nonobstructive azoospermia identified in microarray gene expression analysis Agnieszka Malcher, M.S., Natalia Rozwadowska, Ph.D., Tomasz Stokowy, M.S., Tomasz Kolanowski, M.S., Piotr Jedrzejczak, M.D., Wojmir Zietkowiak, Ph.D., Maciej Kurpisz, M.D. Fertility and Sterility Volume 100, Issue 6, Pages 1686-1694.e7 (December 2013) DOI: 10.1016/j.fertnstert.2013.07.1999 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Hierarchical clustering dendrogram of all samples analyzed according to gene expression profile. Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The box plots represent the genes whose expression was down-regulated in infertile men as compared with the controls. (A) By microarrays. (B) Verified by quantitative PCR. (C) Verified by data obtained from ArrayExpress database. The data from the microarrays were transformed to logarithmic data and are presented in a logarithmic scale, whereas the data from the quantitative PCR are shown as relative expression. Descriptions presented below the box plots demonstrate the fold change value and the P value (the green color indicates statistical significance). Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 The box plots represent the gene expression level undergoing up-regulation in patients who have spermatogenetic maturation arrest at a late stage (subgroup A) compared with the group that has severe spermatogenetic impairment (subgroup B). (A) By microarrays. (B) Verified by quantitative PCR. (C) Verified by data obtained from ArrayExpress database. The data from the microarrays were transformed to logarithmic data and are presented in a logarithmic scale, whereas the data from the quantitative PCR are shown as relative expression. Descriptions presented below the box plots demonstrate the fold change value and P value (the green color indicates statistical significance). Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Western blot analysis of selected protein products. (A) Immunoblots of UBQLN3, FAM71F1, WBSCR28, and ACTB gene products (reference). (B) Relative quantity of proteins normalized to expression level of ACTB. (C) Immunohistochemistry of UBQLN3, FAM71F1, and WBSCR28 gene products in the normal adult human testis. Black arrows indicate a positive signal; white arrows indicate a negative signal. Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Hierarchical clustering dendrogram prepared according to gene expression data profiles obtained from the ArrayExpress database (E-TABM-234). The subgroup A consisted of controls and samples with spermatogenetic failure at postmeiotic stage. The samples with severe failure in spermatogenesis formed subgroup B. Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 Heat map of 4,946 differentially expressed genes (P<.05) in male individuals. Fertility and Sterility 2013 100, 1686-1694.e7DOI: (10.1016/j.fertnstert.2013.07.1999) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions