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Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is a physiological process involved in both normal menstrual cycling and implantation of the embryo. So far, very little is known about the expression of angiopoietins, growth factors involved in angiogenesis, in human endometrium. Both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the endothelial cell-specific receptor tyrosine kinase Tie-2. In this study we determined the mRNA expression of Ang-1, Ang-2 and Tie-2 by quantitative competitive RT/(QC)-PCR (including specifically designed competitor cDNA) in biopsied human endometrium throughout the menstrual cycle. We detected the mRNA for the angiopoietins in 30 out of 32 endometrial biopsies (94%), covering early proliferative (n = 4), mid proliferative (n = 12), late proliferative (n = 3), early secretory (n = 3), mid secretory (n = 5) and late secretory (n = 3) phases. Analysis of the target/competitor ratios (QC-PCR) revealed that Ang-1 mRNA expression was significantly up-regulated (P = 0.027) during the secretory phase of the menstrual cycle. In contrast, the expression levels of both Ang-2 mRNA and Tie-2 mRNA showed only minor variations at different cycle stages. These findings were confirmed by the relative expression ratio of Ang-1 versus Ang-2 in a multiplex PCR. The expression of Ang-1, Ang-2 and Tie-2 mRNA was detected in both isolated endometrial epithelial and stromal cell fractions. Immunohistochemical localization of the proteins revealed qualitative differences in both cell type and cycle stage expression. In conclusion, the enhanced Ang-1 expression during the secretory phase might serve to stabilize the newly developed blood vessels.

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.