This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)
0853733 Marcolongo
Intellectual Merit: One of the critical challenges of modern reproductive biology is to develop viable clinical approaches to treat infertility. Embryo implantation represents a crucial step of the reproductive process and its success relies on the culmination of a well-orchestrated series of spatially and temporally controlled events. The attachment of the blastocyst to the endometrium is mediated, at least in part, by the L-selectin adhesion system. Interactions between L-selectin expressed on the peripheral cells of the human blastocyst, the trophoblasts, and its oligosaccharide ligands on the surface of the human endometrium are thought to provide a "braking" mechanism for the embryo as it enters the endometrial cavity, similar to its role in other biological systems. While the L-selectin mediated interaction is likely not strong enough to sustain attachment (which is further enhanced by secondary integrin-ligand interactions), it is thought to provide strength adequate to allow the blastocyst to be initially captured by the uterine luminal epithelium. However, there is a lack of understanding of the attachment mechanisms associated with primary and secondary adhesion. A principle cause of the 30% failure rates of in vitro fertilization is associated with a defect in the attachment process during implantation; without further understanding of the embryo attachment mechanisms, little can be done to address this critical clinical issue.
There are two objectives of this application: 1) to utilize model cell lines for trophoblasts (Jeg-3) and uterine epithelial cells (Ishikawa) and cell mechanics techniques to examine the dependence of molecular expression (controlled by environmental factors: hormonal stimulation and fluid flow) on primary and secondary attachment strength of the blastocyst/uterine epithelium interactions and 2) to develop and characterize a 3D Jeg-3 trophosphere to better mimic the architecture of the blastocyst and examine biochemical and mechanical interactions of the trophosphere with a 3D tissue engineered structure of human endometrial epithelial cells obtained from biopsies (IRB 15822) to more fully mimic the in vivo environment. The central hypothesis is that the expression and retention of a critical level of L-selectin and L-selectin ligands are necessary to facilitate adequate initial attachment of the blastocyst to the uterine epithelium and similarly the expression of molecules in the integrin-mediated attachment will control the strength of attachment for secondary bonding. The preliminary work has shown that the research team can manipulate L-selectin and L-selectin ligand expression with hormonal conditioning in trophoblast and endometrial epithelial cell models, respectively. For the first time, the attachment strength of a model L-selectin mediated implantation system has been quantified using a parallel plate flow chamber customized with a quartz crystal microbalance (QCM) sensor and functional attachment dependence based on L-selectin expression has been demonstrated. Further, the secondary attachment strength of the model integrin/ligand system has been quantified using a spinning disc apparatus. In addition to our supportive preliminary data, we are particularly poised to undertake this research because an inter-disciplinary team of bioengineers, biochemists and cell biologists and a fertility clinician with the range of skills necessary to answer these critical questions has been assembled.
This project is innovative because it will be the first time that the primary and secondary attachment strengths for blastocyst implantation have been investigated. An additional innovation comes in the use of the 3D trophosphere model to examine the spatial effects that may contribute to the attachment mechanisms. This work also realizes an engineering approach toward challenges of maternal fetal medicine and therefore serves as a milestone at the intersection of these disciplines.
Broader Impacts: Development of the model blastocyst/uterine endometrial epithelial cell system to examine the effect of microenvironment (hormonal and physical) on the primary and secondary attachment mechanisms will allow further research to examine the effects of endogenous factors and possible clinically relevant treatments on the functional behavior of the cells. The 3D models of the trophosphere and the hEEC enable the advancement of the understanding of the effect of the spatial distribution of molecules on attachment. These insights may inspire new treatments for implantation-related fertility, one of the greatest impediments to successful reproductive outcomes in IVF, where, despite fertilization rates of up to 60%, pregnancy rates per transfer of embryos to the uterus are only 42.5%. This program lays the groundwork for exploration of further questions related to infertility that could greatly benefit from an engineering approach. These methods and discoveries will be disseminated to the scientific community through publications and presentations at conferences. The proposed research at the intersection of disciplines will offer excellent training opportunities to students as they study this clinically relevant and important problem.
