Proteomic Analysis of Endometrial Lavage Samples Provides New Insights into Proteins Important for Implantation.

During the mid-secretory phase of the menstrual cycle, under the control of progesterone and oestrogen, increased secretory activity is a characteristic feature of endometrial epithelium. Secretions into the uterine lumen from the endometrium, also known as histotroph, contain important mediators that are postulated to modulate the endometrium in preparation for implantation and to contribute factors essential to the conceptus prior to and during implantation. To date, little is known about these secretions but analysis of their composition would provide a minimally invasive tool to assess the state of the endometrium. Recent advances in proteomic analysis provide the potential to assess the proteins in such biological fluids. The potential clinical applications of this technology are broad-based and include identification of specific proteins, protein profiles, alterations with disease and the development of diagnostic tests. We hypothesized that application of 2D proteomic analysis of human uterine washings would allow identification of proteins important for the establishment of pregnancy in humans. Uterine lavages were collected from (n=6) fertile women during the mid-secretory phase of the menstrual cycle. Preliminary 2D proteomic analysis revealed an abundance of serum proteins, particularly albumin, within the lavage samples. To enrich for low abundance proteins, two methods of serum protein removal were assessed and the protein profile was analyzed over two different pH ranges (separated by their isoelectric points (pI)). Affinity columns that removed the 6 most abundant serum proteins, (multiple affinity removal system (MARS)) provided the most effective approach for enhancing the detection of lower abundant proteins within the uterine lavage. Comparison of depleted and non-depleted fractions using Differential in gel electrophoresis (DiGE) revealed increased numbers of spots detected and the presence of proteins otherwise masked by abundant serum proteins, following MARS treatment. Image analysis and assessment of the protein profile over a wide (pI 3-10) vs. a more narrow pH range (pI 4-7) revealed greater resolution of the protein profile and a further increase in spot number detection over the 4-7 pI range. Protein identification using MALDI-TOF-mass spectrometry revealed a number of novel proteins present in endometrial secretions that are likely to have important roles for conferring receptivity and/or during embryo implantation. Assessment of uterine washings using state-of-the-art proteomics technology provides a useful tool for the identification of the protein repertoire present in the uterine cavity during the crucial peri-implantation stage. Identification of the secreted proteome has many potential applications, including a greater understanding of the proteins present and their roles during embryo implantation, and provision of diagnostic markers for endometrial receptivity tests critical for improving current assisted reproductive technologies.