Estrogen and Progesterone Response Elements in Genes Relevant for Human Embryo Implantation Identified by Microarray/Bioinformatics

Abstract

The process of embryo implantation is a condition of equilibrium in the up- and down-regulation of diverse endometrial genes/proteins under control of steroid hormones and other local factors. The objective of this study was to identify putative estrogen and progesterone response elements (ERE and PRE) in the 5’-flanking regions of a subset of genes found to be differentially expressed in two clinical scenarios: (1) during the transition of the pre-receptive (early luteal phase, day 16) to the receptive endometrium (mid-luteal phase, day 21) in natural cycles; and (2) during the putative window of implantation (day 21) of gonadotropin-stimulated controlled ovarian hyperstimulation (COH) cycle of oocyte donors in GnRH agonist or antagonist cycles compared to temporally-matched natural cycles. Prospective, randomized and blinded clinical research study. RNA was extracted from endometrial specimens; for global gene expression profiling cRNA was generated and hybridized to Affymetrix Hu95A microarrays. Statistical analysis of the genomic data was performed using pair wise multiple group comparison (SAM software). To validate microarray findings we quantified the expression of selected genes using semiquantitative RT-PCR. We retrieved a 3500 bp-long sequence encompassing 3000 bp upstream and 500 bp downstream of the transcriptional start site of each gene. The search for putative EREs was carried out with ERE finder software with a range of sensitivity threshold from 0.75 to 0.87. The presence of potential EREs was also investigated using the MATCH program using nucleotide distribution matrix for ER with core similarity set to the stringency levels optimal to reduce false positives. The search for putative PREs was conducted using a pattern-based method implemented in the GeneQuest module of Lasergene software. (1) Candidate ERE sequences were found in the 5’-flanking regions of 10 genes among 12 genes analyzed that were differentially expressed when comparing the receptive versus the pre-receptive endometrium; 8 of such genes (CD55, SERPING1, ID4, GATA2,MAPK3K5, CIR, MSX1 and GATA 2) had multiple ERE-like sequences. Seven genes had putative PREs. (2) Linear discriminant analysis identified 4 genes (OPTN, SNX7, PCOLN3, and COX 17) significantly up regulated in COH versus natural cycles with maximal potential functional significance. For these genes, the presence of ERE-like sequences was significantly higher than an average for the human genome. Of major interest was the discovery of an identical ERE-like sequence (TGGCCA-NNN-TGGTCT) in the promoter regions of extended homology of these genes. PREs were identified in two genes. This study identified a number of genes that may have functional significance for the opening and maintenance of the window of receptivity. Although induction of expression by steroid hormones has been previously detected for some of these genes, the mechanism of their transcriptional activation remains unknown. It is reasonable, however, to assume that in the endometrium, transcription of the implantation-related genes is directly regulated by nuclear estrogen and/or progesterone receptors. Our results demonstrated that most of these genes depict putative EREs and PREs. The identification of a unique pattern of ERE-like sequence in a subset of genes upregulated during COH cycle provides strong support for their functional significance.