title: Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.
abstract: OBJECTIVE
  Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma?
  
  
  CONCLUSIONS
  The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells.
  
  
  BACKGROUND
  Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle.
  
  
  METHODS
  Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments.
  
  
  METHODS
  Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively.
  
  
  RESULTS
  Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures.
  
  
  CONCLUSIONS
  Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels.
  
  
  CONCLUSIONS
  Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives.
  This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.
authors.affiliation: Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Herestraat 49 box 611, B-3000 Leuven, Belgium.
authors.forename: Katrien
authors.initials: K
authors.lastname: De Clercq
authors.affiliation: Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Herestraat 49 box 611, B-3000 Leuven, Belgium Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), KU Leuven, Herestraat 49 box 802, B-3000 Leuven, Belgium.
authors.forename: Katharina
authors.initials: K
authors.lastname: Held
authors.affiliation: Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Herestraat 49 box 611, B-3000 Leuven, Belgium.
authors.forename: Rieta
authors.initials: R
authors.lastname: Van Bree
authors.affiliation: Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: Christel
authors.initials: C
authors.lastname: Meuleman
authors.affiliation: Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: Karen
authors.initials: K
authors.lastname: Peeraer
authors.affiliation: Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: Carla
authors.initials: C
authors.lastname: Tomassetti
authors.affiliation: Laboratory of Ion Channel Research and TRP Research Platform Leuven (TRPLe), KU Leuven, Herestraat 49 box 802, B-3000 Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Voets
authors.affiliation: Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Gasthuisberg, B-3000 Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: D'Hooghe
authors.affiliation: Laboratory of Obstetrics and Experimental Gynaecology, KU Leuven, Herestraat 49 box 611, B-3000 Leuven, Belgium joris.vriens@med.kuleuven.be.
authors.forename: Joris
authors.initials: J
authors.lastname: Vriens
mesh_terms: D000328:Adult; D003592:Cytophotometry; D004717:Endometrium / Q000166:cytology / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D008183:Luteal Phase; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D017154:Stromal Cells / Q000378:metabolism*; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D050051:Transient Receptor Potential Channels
keywords: Ca2+-permeable ion channels; human endometrium; implantation; stromal cells; transient receptor potential channels