Periodic Reporting for period 1 - EXORep (EXOsomes as a new strategy in Assisted Reproductive Technologies: tracing maternal nanomessengers to improve pregnancy outcomes)
Periodo di rendicontazione: 2020-09-01 al 2022-08-31
Assisted reproductive technologies (ART) have come a long way in the last 30 years both in animals and humans and are well established in the clinical practice. In vitro embryo production (IVP) is performed and transfer of embryos at the blastocyst stage to achieve successful pregnancy. However, even after improvements of the technology, the quality of IVP embryos is still lower compared to the in vivo ones. The lack of embryo-maternal interactions and the maternal signals in the in vitro embryo culture conditions have been postulated as the main reason for the lower embryo quality. Recently, a new mechanism has been proposed pointing at the potential role of extracellular vesicles (EVs) as important mediators of the early embryo-maternal crosstalk. EVs are present in various biofluids and carry different biomolecules such as proteins, nucleic acids (different types of RNAs and DNA), lipids, and metabolites that provide a snapshot of the parental cells at the time of secretion but also transfer specific signaling and regulatory molecules. For example, the EVs’ molecular cargo has been used as biomarkers for diagnosis and prognosis of cancer and other diseases in recent years.
Therefore, in the present project, we hypothesized that the oviductal environment as the first site of interactions between the mother and the early embryo, and particularly the oviductal EVs (oEVs) could be important for optimal embryo development. The main objective of this project was to investigate the potentials of oEVs to improve IVP conditions to achieve better pregnancy rates in dairy cattle, but also in humans. The bovine model was used because of its similarities to human during early embryo development.
To reach this overall objective, the following specific objectives were addressed:
Objective 1. Analysis of oEVs in response to the embryo and its different stages of development (before and after genome activation).
Objective 2. Analysis of oEVs in response to good or poor-quality embryos.
Objective 3. Identification of crucial molecules contained in oEVs involved in the support of embryo development.
Objective 4. Evaluation of the potential ability of oEVs’ to improve ART outcomes.
Therefore, in the present project, we hypothesized that the oviductal environment as the first site of interactions between the mother and the early embryo, and particularly the oviductal EVs (oEVs) could be important for optimal embryo development. The main objective of this project was to investigate the potentials of oEVs to improve IVP conditions to achieve better pregnancy rates in dairy cattle, but also in humans. The bovine model was used because of its similarities to human during early embryo development.
To reach this overall objective, the following specific objectives were addressed:
Objective 1. Analysis of oEVs in response to the embryo and its different stages of development (before and after genome activation).
Objective 2. Analysis of oEVs in response to good or poor-quality embryos.
Objective 3. Identification of crucial molecules contained in oEVs involved in the support of embryo development.
Objective 4. Evaluation of the potential ability of oEVs’ to improve ART outcomes.
The work performed during the project is explained, according to the description of the action (DoA) originally proposed, and any deviation from the original plan has been included.
Experiment 1. Analysis of oEVs derived from bovine oviduct epithelial cells in response to the presence of absence of embryos and their embryo quality (poor vs good embryos)
In vitro co-culture of bovine oviduct epithelial cells (BOEC) with or without good or poor-quality bovine embryos was performed in this experiment. Additionally, embryos and BOEC were cultured alone. This experiment was conducted in collaboration with Dr. Rizos at the National Institute for Agriculture and Food Research and Technology (INIA), Department of Animal Reproduction, Madrid, Spain). Culture media (CM, containing the EVs) and embryos were analyzed in the laboratory at the University of Zurich. The EVs were successfully isolated from the CM and characterized using electron microcopy, Western blot, flow-cytometry, and nanoparticle tracking analysis. Subsequently, small regulatory RNAs, so-called microRNAs (miRNAs) contained in the EVs and embryos were analyzed by RNA-sequencing.
Overall, a total of 84 different miRNAs were identified in EVs and 187 in embryos. An overlap of 64 miRNAs was found between EVs and embryos, with 20 unique for EVs and 123 for embryos. The data analysis showed that the miRNA cargo of the EVs secreted by the BOEC varies depending on the embryo presence or absence. Besides, it showed changes depending on embryo quality. In addition, the miRNA profile of the early embryo is different in co-culture with BOEC or alone. Interestingly, this effect of the cells on the embryo miRNA profile is depending on the embryo quality.
Experiment 2. Bioengineered oEVs: loading EVs with regulatory RNAs to assess their ability for targeted delivery and improvement of assisted reproductive technologies
The idea of this experiment was to load in vitro derived oEVs with important regulatory RNAs and perform a transfer of these molecules via oEVs to BOEC, sperm, and embryos. We selected the miRNA miR-449a and the messenger RNA (mRNA) for OVGP1, which are known for their role in the oviduct during the passage of gametes and embryos, and which are lacking in the in vitro conditions. We developed a successful method for in vitro loading of oEVs with miR-449a and OVGP1 mRNA and showed the transfer into BOEC and sperm. The changes of the expression of selected genes (related to embryonic growth and development, oviduct metabolism, sperm-oviduct binding) in the BOEC after the oEV-mediated uptake of miR-449a indicated the potential of oEV as nanocarriers to improve ART conditions.
Experiment 1. Analysis of oEVs derived from bovine oviduct epithelial cells in response to the presence of absence of embryos and their embryo quality (poor vs good embryos)
In vitro co-culture of bovine oviduct epithelial cells (BOEC) with or without good or poor-quality bovine embryos was performed in this experiment. Additionally, embryos and BOEC were cultured alone. This experiment was conducted in collaboration with Dr. Rizos at the National Institute for Agriculture and Food Research and Technology (INIA), Department of Animal Reproduction, Madrid, Spain). Culture media (CM, containing the EVs) and embryos were analyzed in the laboratory at the University of Zurich. The EVs were successfully isolated from the CM and characterized using electron microcopy, Western blot, flow-cytometry, and nanoparticle tracking analysis. Subsequently, small regulatory RNAs, so-called microRNAs (miRNAs) contained in the EVs and embryos were analyzed by RNA-sequencing.
Overall, a total of 84 different miRNAs were identified in EVs and 187 in embryos. An overlap of 64 miRNAs was found between EVs and embryos, with 20 unique for EVs and 123 for embryos. The data analysis showed that the miRNA cargo of the EVs secreted by the BOEC varies depending on the embryo presence or absence. Besides, it showed changes depending on embryo quality. In addition, the miRNA profile of the early embryo is different in co-culture with BOEC or alone. Interestingly, this effect of the cells on the embryo miRNA profile is depending on the embryo quality.
Experiment 2. Bioengineered oEVs: loading EVs with regulatory RNAs to assess their ability for targeted delivery and improvement of assisted reproductive technologies
The idea of this experiment was to load in vitro derived oEVs with important regulatory RNAs and perform a transfer of these molecules via oEVs to BOEC, sperm, and embryos. We selected the miRNA miR-449a and the messenger RNA (mRNA) for OVGP1, which are known for their role in the oviduct during the passage of gametes and embryos, and which are lacking in the in vitro conditions. We developed a successful method for in vitro loading of oEVs with miR-449a and OVGP1 mRNA and showed the transfer into BOEC and sperm. The changes of the expression of selected genes (related to embryonic growth and development, oviduct metabolism, sperm-oviduct binding) in the BOEC after the oEV-mediated uptake of miR-449a indicated the potential of oEV as nanocarriers to improve ART conditions.
Previous studies have identified oEVs as essential components of the oviductal environment. Their characterization at protein level revealed a set of proteins with potential roles in gamete/embryo oviduct interactions. It has also been demonstrated that oEVs can be taken up by the embryo, enhancing their embryo development and quality. However, it was not very clear how the presence of the embryo and its quality modulates the oEV cargo. This project provides a valuable insight into changes of the oEV miRNA content depending on the presence of good or poor-quality embryos and delivered potential biomarkers indicating embryo quality. This could be used for the development of EVs-based assays of embryo quality that could assist the current morphology-based evaluation of embryo quality, enabling embryologists to select the best embryo for uterine transfer and improve pregnancy outcomes. On the other hand, the successful engineering by loading oEVs with regulatory molecules provides a first step towards improving IVP conditions. The project results could be used by researchers in the field of reproduction and fertility, but also in general for development of new drug delivery systems.