OLIGO-SPERM

Male infertility is an important health concern expected to have a major genetic etiology, however despite the recent identification of numerous genetic factors by high throughput sequencing, its diagnosis efficiency remains disappointingly low. Indeed, while gene defects are identified in more than 50% of rare and severe sperm anomalies, the diagnosis yield of common and moderate forms of male infertility such as oligozoospermia stagnates and do not exceed 10%. As only monogenic Mendelian transmissions are currently investigated, we raise the hypothesis that this low diagnostic efficacy could at least be partly due to a complex etiology, and hypothesize that many of the unsolved cases of male infertility could result from oligogenic events, i.e. the accumulation of several rare hypomorphic variants in distinct, functionally connected genes. Our preliminary results in the mouse show that the accumulation of heterozygous mutations in different genes associated to flagellar defects induce a significant increase of sperm morphological defects and an important reduction of sperm motility. These initial findings suggest that the accumulation of deleterious variants could alter male reproductive fitness and that oligogenic inheritance may be a frequent cause of male infertility. We aim to follow up on these preliminary experiments to confirm the reality of oligogenism in male infertility and to estimate its frequency in men with intermediate sperm defects such as oligozoospermia. Using the CRISPR/Cas 9 technology we will generate multiple-mutant mouse models (MMM) with heterozygous mutations in different genes associated with infertility (WP1). In our preliminary work we mutated multiple genes affecting the same sperm function (flagella assembly and movement). Now we will generate MMM carrying up to five heterozygous mutations targeting genes with a broader function, controlling germ cell proliferation and/or meiosis. A precise reproductive phenotyping will be performed for each MMM with a full sperm analysis to identify the morphological and functional defects induced by these cumulated mutations. In addition, the sperm DNA quality (DNA compaction and fragmentation) and the non-coding RNA payload of MMM will be compared to wild-type mice. Last, the fertilizing and developmental competence of MMM sperm will be assessed in vivo (breeding experiments) but also in vitro (IVF, ICSI). To confirm the importance of oligogenism in human infertility we then propose to investigate the prevalence of oligogenic inheritance by comparing the mutational load in sperm related genes between infertile males and fertile controls (WP2). Using exome sequencing, we will compare the mutational burden of rare coding variants affecting fertility genes between 300 unrelated individuals with idiopathic male infertility associated with a mild or moderate oligozoospermia (>5M/ml and <15M/ml) and 300 fertile men. Patients and controls’ recruitment will be achieved with the collaboration with the BLEFCO and CECOS federations. After variant calling and filtering, we will perform a case-control analysis focusing on deleterious variants affecting a panel of approximately 500 infertility genes (in silico panel) selected after a thorough analysis of all available data (literature, expression, animal model). In addition, we will construct the “spermatogenesis biological network”, by performing an integrative bioinformatics analysis of infertility gene products in order to identify new and less obvious candidate genes for infertility and to improve our understanding of spermatogenesis. Overall, this project should expand our understanding of the genetic basis of male infertility, permit to overcome some diagnostic impasses, lead to a greater accuracy in phenotypic prediction and permit to improve patients’ management and genetic counseling.