Endometrial transcriptome analysis indicates superiority of natural over artificial cycles in recurrent implantation failure patients undergoing frozen embryo transfer  Signe Altmäe, Karin Tamm-Rosenstein, Francisco J. Esteban, Jaak Simm, Liis Kolberg, Hedi Peterson, Madis Metsis, Kai Haldre, José A. Horcajadas, Andres Salumets, Anneli Stavreus-Evers  Reproductive BioMedicine Online  Volume 32, Issue 6, Pages 597-613 (June 2016) DOI: 10.1016/j.rbmo.2016.03.004 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Cluster analysis of Z-scored gene expression values in the endometrium at the window of implantation in healthy women with proven fertility in natural cycles (NC-FC), in infertile women undergoing a natural cyle for frozen embryo transfer (NC-FET), and infertile women undergoing an artificial cycle for frozen embryo transfer (AC-FET). Red represents genes with a positive Z score and green, genes with negative Z score. Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 The three differentially expressed gene (DEG) groups analysed: DEGs whose expression improved with artificial cycle in infertile women (n = 620) (i.e. DEGs unique for women who have experienced recurrent implantation failure undergoing a natural cycle (NC-RIF) versus healthy women with proven fertility in natural cycles (NC-FC) comparison: meaning that the initial gene expression profile in NC-RIF women differed from that in fertile controls, whereas these DEGs in the AC-RIF (women who have experienced recurrent implantation failure undergoing an artificial cycle) group versus NC-FC comparison demonstrated similar values to fertile controls); DEGs whose expression profile deteriorated with artificial endometrium preparation in infertile women (n = 640) (i.e. DEGs unique for AC-RIF versus NC-FC comparison: meaning that in the AC-RIF versus natural NC-FC comparison the gene expression profile was different, whereas the comparison of gene expression profiles in natural NC-RIF versus NC-FC demonstrated similar values); and genes specific to RIF, n = 269 (i.e. DEGs whose expression was significantly different in infertile women in both natural cycle and artificial cycle when compared with fertile controls). Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Functional enrichment analysis of the endometrial transcriptome at the window of implantation: genes – whose expression improved with artificial cycles (A), genes whose expression deteriorated with artificial cycles (B), and genes specific to recurrent implantation failure (C). Bar colour denotes different types of evidence from gene ontology (DAVID) and pathway (IPA) databases: BP, biological process (in orange); CC, cellular component (in red); CP, canonical pathways (in blue); MF, molecular function (in green). The X-axis denotes functional enrichment score, computed as −log2 of related P-values. Some abbreviations in the Y-axis are indicated in full text in Supplementary Tables S2–S4. Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Tukey test for pairwise comparison of oestrogen receptor elements and progesterone receptor element profiles in promoter regions (−50,000 bp to +150 bp) of differentially expressed genes (DEGs) with artificial cycle deteriorated (green), artificial cycle improved (yellow) and recurrent implantation failure (RIF) specific (red). Box plot showing median and mean value. On the left side significant difference of the presence of oestrogen receptor element sites upstream of DEGs artificial cycle deteriorated compared with artificial cycle improved (P = 3.2e−06) and compared with RIF-specific genes (=0.0045) is shown. On the right significant difference of the presence of progesterone response element sites upstream of DEGs artificial cycle deteriorated compared with artificial cycle improved (P = 0.0052) is shown. AC, artificial cycle; ERE, oestrogen response element; HRE, hormone response element; GRE, glucocorticoid response element; PRE, progesterone response element; RIF, recurrent implantation failure. Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 Two most prevalent hormone response elements found in promoter regions among differentially expressed genes identified in this study. Letters abbreviate the nucleotides (A, C, G, and T) in the images and are sized according to their relative occurrence. (A) ESR1 element (V$ESR1_01) was over represented upstream of differentially expressed genes (DEGs) deteriorated with artificial cycles (examples IL9R, MMP17, PTGER3, ESR2, and GATA3). This oestrogen receptor element motif in the most proximal promoter region (from −1,000 bp to +150 bp from transcription start sites) turned out to be significantly overrepresented in upstream of DEGs that deteriorated with artificial cycle compared with DEGs that improved with artificial cycle (P = 0.023); (B) progesterone response element (V$PR_Q6) was statistically more frequent in upstream of genes related to recurrent implantation failure (examples AFM, BRINP3, CNNM1, FAM151A, and IL12RB2) (P = 0.04) and among DEGs that improved with artificial cycle (examples ADAD1, CALML5, FAM196A, IFNA5, and IL21) (P = 0.04). Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 6 Genes regulated by oestradiol and progesterone in endometrium and Ishikawa cells. Fifty differentially expressed genes (DEGs) from the present study were also regulated by oestradiol and progesterone in our previous study using Ishikawa cells (Tamm-Rosenstein et al., 2013). Red denotes up-regulation and green down-regulation of gene expression in Ishikawa cells. The dotted line indicates zero and the variable line shows the expression change in response to hormone treatment (E2) – gene expression regulated by oestradiol; (P4) gene regulated by progesterone, and (E2, P4) – gene regulated by both oestradiol and progesterone. *Designates genes in the present study with an opposite expression direction versus Ishikawa cells. Red on the y-axis indicates genes whose expression improved with artificial cycle, green indicates genes whose expression deteriorated with artificial cycle, and blue highlights genes specific to recurrent implantation failure. Reproductive BioMedicine Online 2016 32, 597-613DOI: (10.1016/j.rbmo.2016.03.004) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions