Placental Growth Factor Is Secreted by the Human Endometrium and Has Potential Important Functions during Embryo Development and Implantation

Abstract

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the mid-secretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen provides important mediators that modulate the endometrium and support the conceptus during implantation. Previously we demonstrated the importance of vascular endothelial growth factor (VEGF) in the human uterus, particularly with respect to embryo implantation. In the current study, proteomic analysis of human uterine lavage fluid identified the presence of placental growth factor (PlGF) a homolog of VEGF, that binds the VEGF receptor 1 (VEGFR1). Analysis of immunostaining for PlGF in human endometrial tissue across the menstrual cycle (from both fertile and infertile women) revealed PlGF was predominantly localised to glandular and luminal epithelial cells, with staining in the decidualising stromal cells surrounding the maternal spiral arteries in the secretory phase of the menstrual cycle. Immunoreactive PlGF was also detected in subpopulations of endometrial leukocytes. Functional studies demonstrated that culturing mouse embryos with recombinant human (rh)PlGF enhanced blastocyst cell number and outgrowth. Furthermore, treatment of human endometrial epithelial cells (EEC) with rhPlGF enhanced EEC adhesion. Taken together, these data demonstrate that PlGF is abundant in the human endometrium, and secreted into the uterine lumen where it mediates functional changes in cellular adhesion with important roles in implantation.

Conflict of interest statement

DKG receives grant support from Vitrolife AB. This funding does not cover any work presented here. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Immunohistochemical localization of Placental Growth Factor (PlGF) in human endometrium.

Proliferative phase endometrium showing diffuse weak immunostaining for PlGF in glandular epithelium (GE) and leukocytes (Δ)(A). More intense staining was seen in the early-secretory (B), mid-secretory (C) and late-secretory (D) phases. Immunoreactive staining was localized in basal luminal ‘vesicles’ of the GE in the early-secretory phase (B) these immunoreactive vesicles were apically located in the mid-secretory (D) and late secretory (F) phase, as indicated by arrowheads. Immunoreactive protein was also detected in blood vessels and decidualising stroma surrounding the spiral arterioles (C & E; indicated by arrows). (Endometrial biopsies; n = 5–7 samples/phase of the cycle). No staining was observed in the negative control (isotype matched IgG). Scale bar on A and E = 100 μm, B and C = 200μm, D and F = 50μm.

Fig 2. Effects of recombinant human (rh)PlGF on mouse embryo development in culture.

Mouse embryos cultured in the presence of 5 and 50ng/mL of rhPlGF had significantly more cells on day 5 of culture than controls. Embryos cultured in the presence of PlGF at a dose of 500ng/mL had no increase in blastocyst cell number. Representative blastocyst images are shown where cell nuclei can be seen (blue) stained with Hoechst. Data are expressed as mean ± SEM. (n = 24-30/treatment group). Data was statistically analyzed by analysis of variance followed by Dunnett's multiple comparisons test (A). Blastocyst outgrowth (on fibronectin 10μg/ml) was significantly enhanced at 46, 65, 72, 89, 96 and 113hrs when embryos were cultured in the presence of rhPlGF (50 and 500 pg/mL) compared to control (B). Blastocysts cultured in PlGF at a dose of 5ng/mL did not exhibit outgrowth on fibronectin. Representative images are shown where blastocyst outgrowth can be visualised (at 113 h) at different doses of PlGF. 5 = 5ng/mL; 50 = 50ng/mL and 500 = 500ng/mL of rhPlGF). (n = 15 embryos/treatment). ‘a’ represents the 50pg/mL dose where p

Fig 3. Endometrial epithelial cell adhesion to the extracellular matrix component fibronectin.

Treatment with 50ng/ml rhPlGF significantly enhanced adhesion of endometrial epithelial cells to fibronectin in real time (A, p