OSTEOPONTIN AND ITS RECEPTORS AT THE EPITHELIAL-EMBRYO INTERFACE AT IMPLANTATION

Abstract

Failure of implantation is a high-prevalence cause of human infertility. Implantation is a cascade of morphological and molecular events from the initial attachment of an embryo to the endometrial epithelium to its invasion into the underlying tissue. One of the main reasons for the stagnant success rate of assisted reproductive technologies is the scarce knowledge about the process of embryo implantation and its regulation. Therefore, a more detailed understanding of the molecular mechanism is required to help address broader issues in human reproduction. To overcome the lack of in vivo accessibility, an in vitro model was used comprising co-culture of endometrial epithelial (Ishikawa) cells with either human or mouse preimplantation embryos for 48 hours. The model was refined to enable the attachment kinetics, stability and outgrowth of embryos to be analysed. Mouse embryo attachment to Ishikawa cells occurred weakly during the first 24 hours and then became stable from 28 hours. Moreover, 80% of embryos were outgrowing at 48 hours. Despite the weak adhesion during the first 24 hours, the absence of coculture for this period of the experiment was detrimental to embryo outgrowth. Assisted hatching of both mouse and human embryos was identified as essential before attachment could be initiated. Osteopontin (OPN) was hypothesised to play a role in embryo adhesion via its receptors alphaνβ3 integrin and CD44. All these components are present at the cell membrane in both trophectoderm and endometrial epithelium at the time of implantation. These data set the scene for functional investigation of their involvement in implantation. Binding of OPN to its endometrial receptors was altered by the addition of function/binding-blocking antibodies, RGD hexapeptide or recombinant human OPN. Blocking the binding site occupancy of alphaνβ3 integrin using antibodies slowed mouse embryo attachment by up to 8 hours. A functionblocking antibody against CD44 induced a 12-hour delay in the stabilisation of attachment. Despite the delay in the attachment kinetic observed in the presence of function-blocking antibodies, only the addition of OPN or RGD hexapeptide impaired embryo attachment and outgrowth; a decrease in the number of outgrowing embryos also resulted (both) and in the area of outgrowth (RGD peptide only). Pretreatment of endometrial cells with hyaluronidase (HYAL2) improved embryo attachment and its stabilisation. Association between insulin-like growth factor 1 receptor (IGF1R) and alphaνβ3 integrin was demonstrated, and IGF-I supplementation increased extracellular exposure of OPN. Therefore, the effect of IGF-I on embryo attachment and outgrowth was investigated in co-culture. However, no change in the attachment kinetics was noted. Therefore, the role of IGF-I in embryo adhesion remains elusive. These experimental studies demonstrate the involvement of OPN in embryo attachment and reveal a novel function for CD44. Further investigations will be required to discriminate the role of OPN- and HA-CD44 interactions at implantation, which have potential treatment implications for assisted reproduction.

Date of Award	19 Jul 2016
Original language	English
Awarding Institution	The University of Manchester
Supervisor	John Aplin (Supervisor), Susan Kimber (Supervisor) & Melissa Westwood (Supervisor)