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Title: The role of matrix metalloproteinases and relaxin hormone in trophoblast-endometrium interactions during implantation
Author: Sarb, Jamela
ISNI:       0000 0004 7226 7759
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Several modulatory molecules interact at the trophoblast-endometrial interface to generate a favourable environment for blastocyst implantation. Successful implantation requires a delicate balance between the tissue inhibitor matrix metalloproteinases (TIMPs) and activators of matrix metalloproteinases (MMPs) which are involved in the synthesis and degradation of extracellular matrix. The tissue hormone relaxin (RLX) has been reported to stimulate MMPs activity in various species including human, pig and rat. In this study we hypothesised that trophoblast-endometrial interactions are influenced by the functional expression of MMPs, TIMPs and human RLX during early implantation. The aims of the study are to determine whether RLX-2, MMPs and TIMPs levels would be clinically useful predictive markers of impending pregnancy loss, or other adverse pregnancy outcomes, and to explore the trophoblast-endometrial interactions under the influence of altered expression levels of RLX-2, MMP-2 and TIMP-2. We conducted both in vivo and in vitro studies. In vivo: Four cohorts of pregnant women were recruited. These were a) pregnant women with a viable pregnancy and a history of recurrent miscarriage (RM), b) pregnant women, matched for gestational age with no history of RM (as controls), c) women seen in early pregnancy presenting with threatened miscarriage and d) healthy controls also matched for gestational age. Serum RLX-2, MMP-2, MMP-9 and TIMP-2 levels were determined in each of these cohorts by ELISA and compared for corresponding study gestations. In vitro: studies involved co-incubation of T-HESCs and JAR cell lines with increasing concentrations of RLX-2, TIMP-2 and Batimastat to the cultures, Production of MMP-2 in the supernatant was evaluated by ELISA. In vitro 2D and 3D endometrial models, and JAR spheroids were developed and employed to study attachment and invasion stages of implantation and determine how these processes were affected by MMP-2, MMP-9, TIMP-2 and RLX-2. Results: In vivo study: There was a significant increase in the serum expression of TIMP-2, and a significant decreased in the serum expression of MMP-2, MMP-2/TIMP-2 complex and RLX-2 hormone in RM compared to their controls with no history of RM. In vitro study: Stimulation of T-HESCs with RLX-2 hormone showed increased secretion of TIMP-2. The data also showed that RLX-2 increased MMP-2 expression by JAR trophoblast cells in a dose-dependent manner, with significant differences being observed compared to unstimulated cells. Cultured T-HESCs and JAR with TIMP-2 showed a biphasic effect. Both showed decreased levels of MMP-2 in the medium after 48h in comparison to unstimulated controls, this was followed by increase in MMP-2 levels compared to the day before. While the effect Batimastat on the levels of secreted MMP-2 by both T-HESCs and JAR cells showed a dramatic decline in secreted MMP-2 levels in both a time- and dose-dependent manner. The kinetics of attachment between endometrial epithelial and/or endometrial stromal monolayers with the JAR spheroids under the effect of RLX-2, MMP2, MMP-9 and TIMP-2 co-incubation indicated that TIMP-2 significantly decreased the attachment rate of JAR spheroids to endometrial stromal cell line in a time-dependent manner of co-incubation. The expansion of the trophoblast cells over the endometrial stromal cells was promoted mainly by MMP-2 and inhibited by a Batimastat inhibitor. It was surprising that the Batimastat inhibitor showed a significant decrease in expansion whereas recombinant TIMP-2 did not. In conclusion, it appears that MMP-2, TIMP-2 and RLX-2 hormone among other proteins play a key role in early human embryo implantation stages. The in vivo data suggest that any dysregulation of any of these proteins may play a role in the pathogenesis of implantation and recurrent pregnancy loss. The in vitro data also suggests that MMP-2 and TIMP-2 appear to be implicated in this process. However, there is a need to optimise a cell culture model to promote and assist in understanding the potential mechanism and the signalling pathways of trophoblast attachment and invasion during implantation. Such a model could provide a platform for further studies to reduce implantation failure and recurrent pregnancy, thereby improving pregnancy success rate following both spontaneous and assisted conception.
Supervisor: Anumba, Dilly ; Murdoch, Craig Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available