Genomic and proteomic analyses of the endometrium during the window of implantation following laser capture microdissection: Comparison of natural and controlled ovarian hyperstimulation (COH) cycles

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 compare endometrial gene and protein expression during the window of implantation of natural and COH cycles following tissue compartmentalization using laser capture microdissection (LCM). Prospective, randomized and blinded research study. Endometrial biopsies were obtained during the putative window of implantation (day 21) from (a) COH cycles of oocyte donors (n = 10); and (b) natural cycle controls (n = 5). Oocyte donors were stimulated either with a GnRH agonist (long protocol) and recombinant FSH (rFSH) or with rFSH and a GnRH antagonist. For global gene expression profiling, cRNA was generated and hybridized to Affymetrix Hu95A microarrays. Prior to proteomic analysis, LCM was used to isolate specific endometrial cell types, i.e. epithelial and stromal cells. Proteomics was performed to the compartmentalized endometrium using surface enhanced laser desorption/ionization (SELDI)-time of flight spectrometry (TOF) technology. Protein lysates were loaded robotically on an IMAC 3 chip pretreated with CuSO4. Each resulting protein peak was determined and its intensity normalized for total ion current. Statistical analysis of the genomic data was performed using pair wise multiple group comparison (SAM software), while statistical analysis of the proteomic data was based on the Student’s t test and CART analysis using Ciphergen’s Biomarker Patterns Software. SAM analysis reveled that the endometrium of COH cycles differed in 18 genes in comparison to the timely matched natural cycles. Optineurin and procollagen type 3 endopeptidase were genes up regulated in the COH group, which were validated by real time RT-PCR. SELDI spectra revealed similar protein fingerprinting in the dissected endometrial epithelium in natural versus COH cycles stimulated with a GnRH antagonist. On the other hand, microdissected epithelium from GnRH agonist cycles depicted a different fingerprinting compared to natural and GnRH antagonist cycles. Furthermore, microdissected stromal compartments differed in the protein fingerprinting in the three studied groups. Several proteins, with masses ranging from 2.5 to 17 kD were differentially expressed among the three groups. Importantly, two unique 11 kD and 15 kD protein peaks were observed in the GnRH agonist cycles. Minimal differences in gene expression were found when comparing COH versus natural cycles. SELDI fingerprinting revealed a similar protein expression pattern in the epithelium of natural and GnRH antagonist cycles. However, differences were observed in GnRH agonist cycles. The stromal compartment demonstrated divergent protein fingerprinting amongst groups. These results provide a basic background on global gene and protein expression analysis of the endometrium during the window of implantation under natural and stimulated conditions.