Granulocyte-Colony Stimulating Factor Related Pathways Tested on an Endometrial Ex-Vivo Model Mona Rahmati 1,2 *, Marie Petitbarat 1 , Sylvie Dubanchet 1 , Armand Bensussan 1 , Gerard Chaouat 1 , Nathalie Ledee 1,2 1 Equipe ‘‘Implantation et Dialogue Cytokinique Me `re-Conceptus’’, UMRS-976, INSERM, Universite ´ Paris Diderot, Hopital Saint Louis, Paris, France, 2 Service d9Assistance Medicale a la Procreation, Hopital Pierre Rouques - Les Bluets, Paris, France Abstract Introduction: Recombinant human Granulocyte-Colony Stimulating Factor (rhG-CSF) supplementation seems to be a promising innovative therapy in reproductive medicine, used in case of recurrent miscarriage, embryo implantation failure or thin endometrium, although its mechanisms of action remain unknown. Our aim was to identify possible endometrial pathways influenced by rhG-CSF. Materials and Methods: Hypothetical molecular interactions regulated by G-CSF were designed through a previous large scale endometrial microarray study. The variation of endometrial expression of selected target genes was confirmed in control and infertile patients. G-CSF supplementation influence on these targets was tested on an endometrial ex-vivo culture. Middle luteal phase endometrial biopsies were cultured on collagen sponge with or without rhG-CSF supplementation during 3 consecutive days. Variations of endometrial mRNA expression for the selected targets were studied by RT-PCR. Results: At the highest dose of rhG-CSF stimulation, the mRNA expression of these selected target genes was significantly increased if compared with their expression without addition of rhG-CSF. The selected targets were G-CSF Receptor (G- CSFR), Integrin alpha-V/beta-3 (ITGB3) implicated in cell migration and embryo implantation, Plasminogen Activator Urokinase Receptor (PLAUR) described as interacting with integrins and implicated in cell migration, Thymidine Phosphorylase (TYMP) implicated in local angiogenesis, CD40 and its ligand CD40L involved in cell proliferation control. Conclusion: RhG-CSF seems able to influence endometrial expressions crucial for implantation process involving endometrial vascular remodelling, local immune modulation and cellular adhesion pathways. These variations observed in an ex-vivo model should be tested in-vivo. The strict indications or counter indication of rhG-CSF supplementation in reproductive field are not yet established, while the safety of its administration in early pregnancy on early embryogenesis still needs to be demonstrated. Nevertheless, rhG-CSF appears as a promising therapy in some difficult and unsolved cases of reproductive failure. Indications of pre-conceptual rhG-CSF supplementation may derive from a diagnosed lack of endometrial expression of some target genes. Citation: Rahmati M, Petitbarat M, Dubanchet S, Bensussan A, Chaouat G, et al. (2014) Granulocyte-Colony Stimulating Factor Related Pathways Tested on an Endometrial Ex-Vivo Model. PLoS ONE 9(10): e102286. doi:10.1371/journal.pone.0102286 Editor: Ana Claudia Zenclussen, Medical Faculty, Otto-von-Guericke University Magdeburg, Medical Faculty, Germany Received April 16, 2014; Accepted June 16, 2014; Published October 2, 2014 Copyright: ß 2014 Rahmati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work is supported by Grant for Fertility Innovation, 2010, Merck Serono, http://www.grantforfertilityinnovation.com/en/gfi2010/winners/dr_ nathalie_ledee.html. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: Grant for Fertility Innovation, 2010, Merck Serono. http://www.grantforfertilityinnovation.com/en/gfi2010/winners/dr_nathalie_ledee.html. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials. * Email: mona.rahmati@gmail.com Introduction Recombinant Human Granulocyte-Colony Stimulating Factor (rhG-CSF) is used since late 1980’s in haematological indications such as chemotherapy induced neutropenia [1], congenital agranulocytosis [2] or haematopoietic stem cell transplantation [3]. In reproductive medicine, rhG-CSF supplementation seems to be one of the most promising innovative therapies. Indeed, in distinct countries, rhG-CSF supplementation, either systemic (subcutaneous administration) or local (intra uterine infusion), is evaluated in the context of some unexplained recurrent miscar- riages, repeated embryo implantation failures or thin unresponsive endometrium. Two randomised studies using rhG-CSF supple- mentation on IVF stimulation protocols, in case of repeated miscarriages, suggest a higher live birth rate and fewer cases of pregnancy loss [4] [5]. Moreover, rhG-CSF supplementation is tested in preliminary IVF protocols involving patients with a history of repeated embryo implantation failures (IF) [6]. There are also cases of pregnancy reported after intra-uterine infusion of PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e102286