27 - 30 May 2005

A FUNCTIONAL GENOMICS APPROACH TO ELUCIDATE PARACRINE INTERACTIONS BETWEEN THE HUMAN TROPHOBLAST AND MATERNAL DECIDUA Hess AP [1,2], Hamilton AE [1], Dosiou C [1], Talbi S [1], Nyegaard M [1], Gembacev-Krtolica O [3], Kruessel JS [2], Fisher SJ [3], Giudice LC [1] [1] Department of OB/GYN Stanford University School of Medicine Stanford, CA, USA [2] Department of OB/GYN, Heinrich-Heine University Duesseldorf, Germany [3] Department of Stomatology University of California San Francisco, San Fransisco, CA, USA

Introduction:
Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation throughout early pregnancy. Trophoblasts secrete regulatory products that support their invasion into the maternal endometrium and to establish the placental bed. Global cross-talk between these two tissues, to date, has not been elucidated.
Aim: Design of an in vitro model to examine the gene regulation of decidualized stromal cells (dES) after co-incubation with human trophoblast conditioned media (TCM) to gain a better understanding of the process of implantation.

Material and Methods:
Human endometrial stromal cells were decidualized using progesterone after estradiol priming (using IGFBP-1 as a marker of decidualization). After 14 days of hormonal treatment, cells were further treated with TCM or, as a control, with conditioned media from non decidualized stromal cells (CCM). Decidualized endometrial stromal cells (dES) were harvested after 0, 3 and 12 hr, and total RNA was isolated. cDNA was synthesized and further derived biotinylated cRNA was hybridized to a high density oligonucleotide GeneChip Array (HG-U133 plus 2.0, Affymetrix). The resulting data were analyzed by bioinformatics approaches, and validation of results was performed by real-time RT-PCR and also Western blot analysis.

Results:
The analysis of the GeneChip Arrays showed that 468 genes were significantly up-regulated, after co-incubation of dES with TCM, compared to CCM of which 162 were ESTs. Also, 680 genes were significantly down-regulated, of which 326 were ESTs. Among the most up-regulated genes were chemokines (CXCL 1) and their receptors (CXCR4), interleukins (IL-8), genes involved in the immune response (SCYA8, PTX3) and in membrane stabilization (TNFAIP6, TNFAIP3), and metalloproteinases (MMP1, MMP10, MMP14). Among the down regulated genes were growth factors , e.g., IGF-1, FGF-1, TGFb1 and angiopoietin-1, as well as genes involved in Wnt signaling (Wnt4, FZD2). Real-time RT- PCR as well as Western Blot analysis, confirmed those data for representative genes of both the up-and down regulated groups.

Conclusion:
The data demonstrate a significant up-regulation of genes involved in angiogenesis and initiation of the immune response in decidualized endometrial stromal cells exposed to TCM, suggesting that the trophoblast acts to alter the local immune environment of the endometrium to enhance the process of implantation. Down-regulation of growth factors, (e.g., IGFs and angiopoietin-1 in decidualized endometrial stromal cells exposed to TCM, further suggests a balance to prevent unopposed invasiveness of the trophoblast.

Supported by the NIH Specialized Cooperative Centers Program in Reproduction Research (NIH U-01 HD42298) to LCG and by the German Research Foundation (DFG, HE-3544/1) to APH.

List of abstracts from the 3rd International Conference on the Female Reproductive Tract