Assessment of in vitro impact of low molecular weight heparin on expression of heparanase and heparin binding growth

Abstract

Heparin has a beneficial effect in the treatment of recurrent miscarriages and positively affects implantation rates in the IVF procedure in women with reproductive disorders not associated with thrombophilia. Several studies have indicated that heparin, by blocking the enzymatic activity of heparanase, may affect the structure and function of the extracellular matrix (ECM) and related growth factors. Disturbances in the remodeling (ECM) are believed to be the potential cause of implantation disorders and recurrent miscarriages. Objectives: The aim of the study was the evaluation, on an in vitro model, of the effect of low molecular weight heparin (LMWH) on the expression of heparanase (HPSE) and, important for successful implantation and invasion of trophoblast, heparan sulfate (HS) - binding growth factors, i.e. heparin-binding epidermal growth factor-like (HB-EGF), vascular endothelial growth factor (VEGF-A), fibroblast growth factor (FGF2) in the endometrium, during the implantation window in women with recurrent miscarriage. Method: Biopsy samples, obtained from 10 patients with two or more unexplained miscarriages, were used to construct a co-culture of glandular epithelial cells and stroma. Endometrium in vitro model was supplemented with steroid hormones and enoxaparin 5ug/ml. Using the qPCR, we assessed relative levels of the HPSE, HB-EGF, VEGF-A and FGF2 transcripts in glandular epithelium and stroma in cell culture. Using ELISA, we measured concentrations of the mentioned above factors in culture medium. Results: A statistically significant increase in the relative level of HPSE, HB-EGF, VEGF-A, FGF2 transcripts in the cells of the glandular epithelium and stroma (p<0.001), as well as their increased concentration in the medium of cultures treated with steroid hormones (p<0.001) were observed. However, we found no effect of LMWH supplementation on the level of the investigated factors. Conclusions: Our results show that the importance of the HPSE hydrolytic activity in the endometrium, during the implantation window, may have a secondary function, and/or that beneficial effects of LMWH in women with impaired reproduction have no significant, direct connection with the, catalyzed by HPSE, reconstruction of the ECM and with release of heparin-binding growth factors.