| Summary: |
Reproductive failure and its prevention are aspects that should be considered in the study of human reproduction. Gametogenesis, embryo implantation, placental and embryo-fetal development are events required for a successful conception of a new human being. Epigenetic reprogramming, which occurs during oogenesis, is crucial for oocyte development, maintenance of genomic stability and establishment of epigenetic marks, such as DNA methylation and DNA hydroxymethylation. DNA methyltransferases (DNMTs) are responsible for DNA methylation, the addition of a methyl group to the 5th carbon of cytosine base, resulting in 5-methylcytosine (5mC). DNA hydroxymethylation, which arises from previously methylated DNA consists on the oxidation of 5mC into 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes. Oocyte quality is a key factor for the fertilization process and ulterior embryo development, as the oocyte carries genetic and epigenetic information essential for reproductive success. The interaction between oocytes and the adjacent somatic cells, the cumulus cells, allows the exchange of nutrients, signaling molecules and mRNAS via transzonal projections and gap junctions, supporting oocyte maturation. Normal oocyte development has a crucial role in reproductive success, however other events during embryo-fetal development are also fundamental to a successful pregnancy outcome. Approximately 10% to 15% of clinically recognized pregnancies terminate spontaneously and about 1 to 5% of couples suffer recurrent abortions. Pregnancy loss has a heterogeneous etiology, which results from several known risk factors; however, approximately 50% of this portion of recurrent abortions have an unexplained cause and remain idiopathic. The main aim of this project is to evaluate genetic and epigenetic mechanisms underlying oogenesis and embryo-fetal development, in order to unveil new molecular paths to answer critical questions of reproductive medicine and i  |
Summary
Reproductive failure and its prevention are aspects that should be considered in the study of human reproduction. Gametogenesis, embryo implantation, placental and embryo-fetal development are events required for a successful conception of a new human being. Epigenetic reprogramming, which occurs during oogenesis, is crucial for oocyte development, maintenance of genomic stability and establishment of epigenetic marks, such as DNA methylation and DNA hydroxymethylation. DNA methyltransferases (DNMTs) are responsible for DNA methylation, the addition of a methyl group to the 5th carbon of cytosine base, resulting in 5-methylcytosine (5mC). DNA hydroxymethylation, which arises from previously methylated DNA consists on the oxidation of 5mC into 5-hydroxymethylcytosine (5hmC) by the Ten-eleven-translocation (TET) enzymes. Oocyte quality is a key factor for the fertilization process and ulterior embryo development, as the oocyte carries genetic and epigenetic information essential for reproductive success. The interaction between oocytes and the adjacent somatic cells, the cumulus cells, allows the exchange of nutrients, signaling molecules and mRNAS via transzonal projections and gap junctions, supporting oocyte maturation. Normal oocyte development has a crucial role in reproductive success, however other events during embryo-fetal development are also fundamental to a successful pregnancy outcome. Approximately 10% to 15% of clinically recognized pregnancies terminate spontaneously and about 1 to 5% of couples suffer recurrent abortions. Pregnancy loss has a heterogeneous etiology, which results from several known risk factors; however, approximately 50% of this portion of recurrent abortions have an unexplained cause and remain idiopathic. The main aim of this project is to evaluate genetic and epigenetic mechanisms underlying oogenesis and embryo-fetal development, in order to unveil new molecular paths to answer critical questions of reproductive medicine and improving genetic follow-up during pregnancy. Firstly, we will evaluate critical features associated with
transcriptional regulation and interactions of human oocytes and the surrounding somatic cells during maturation, by transcriptome analysis (RNA-Seq) in both types of cells. Then, the expression of crucial genes and epigenetic regulators genes - DNMTs and TETs- will be evaluated in more samples using quantitative real-time PCR (RT-qPCR). The expression of these epigenetic regulators will be compared with the global levels of 5mC, 5hmC, DNMTs and TETs enzymes by immunofluorescence assay, allowing a more thorough understanding of epigenetic reprogramming during oogenesis. We will also evaluate whether it is possible to use cumulus cells to infer the oocyte transcriptome and epigenetic state, allowing the identification of biomarkers in cumulus cells and the use of the respective oocytes for reproductive treatments. Afterwards, we propose an extensive study of the transcriptome in placental samples from idiopathic pregnancy losses over the three trimesters of gestation, also by RNA-Seq. Subsequently, we will confirm these results in a larger number of placental samples and we will analyse the transcript levels of relevant genes in RNA extracted from matched fetal tissue, by RT-qPCR. Furthermore, the 5mC and 5hmC levels on the regulatory regions of these genes will be evaluated (by targeted bisulfite and oxidative-bisulfite sequencing) and correlated with the expression variations that will be observed.
Lastly, we propose to perform Drop-Seq (10X Genomics), which enables large-scale, highly parallel single-cell transcriptomics, allowing to cluster different cell populations and therefore determine which type of cells in the placenta are contributing to the observed changes in the gene expression. The future findings of this study may uncover new causes underlying idiopathic pregnancy losses and may contribute to improve the genetic counselling offered to couples that experience recurrent pregnancy losses. Additionally, therapies targeting epigenetic mechanisms could be envisaged to be offered to these couples, such as pregnancy treatment with dietary methyl group donors such as methionine, folate, betaine or choline. Therefore, the study of the (epi)genetic factors that influence oocyte development and, potentially, explain some idiopathic pregnancy losses will allow the development of new approaches to help achieve a successful pregnancy. |