mRNAs and small non-coding RNAs in highly purified spermatozoa and seminal plasma of idiopathic infertile men

• Applicant: Dr. Andrej-Nikolai Spiess
• Subject Area: Reproductive Medicine, Urology
• Term: from 2012 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 214733733

The prognosis of the fertility outcome in assisted reproduction technologies (ART) is not reliable when based solely on spermiogram parameters. Consequently, several studies have been conducted in recent years, in which gene expression patterns of human ejaculates were correlated with ART outcome. In our funded project, we employed ejaculates from an in-vitro fertilization program to investigate differential gene expression for different outcomes (pregnancy, no pregnancy, no fertilization of oocyte). To increase statistical power, we used a special data aggregation technique to include and re-analyze four published gene expression data sets. The subsequently filtered differential genes are amenable for validation. Our re-analysis showed that all published gene expression data sets for ART outcome prognosis are massively contaminated with somatic transcripts, an observation that we explain by an insufficient elimination of "round cells" (leukocytes, macrophages, epithelial cells) prior to gene expression analysis. A putative fertility-modulating binding/incorporation of somatic transcripts on or in spermatozoa will be investigated by fluorescence in-situ hybridization. An RNA-sequencing based analysis of fertile and idiopathic infertile men presenting with normozoospermia will be conducted in order to uncover differential expression in idiopathic infertility with concomitant normal spermiogram parameters. Preliminary results indicate a potential role of specific and highly expressed piRNAs for spermatozoal function. In addition, we will sequence the small RNAs of the corresponding seminal plasma to uncover a possible transfer of transcripts between the cellular and seminalplasmatic fraction.It is evident that a strong gene expression heterogeneity of a spermatozoal population may render the identification of fertility-relevant transcripts near impossible. A potential remedy for this problem may be found in the gene expression analysis of single sperm in a high-throughput fashion to uncover a putative heterogeneous distribution of fertility-relevant transcripts in a spermatozoal population. In cooperation with the Institute for Microsystems Technology (IMTEK, Freiburg) we have optimized the protocol of their developed single cell printer as to enable the high-throughput deposition of single spermatozoa ("sperm printing") in microtitre plates. A downstream quantitative real-time PCR on known or from our differential analysis obtained transcripts will serve as a proof-of-principle to reveal a heterogeneous distribution of transcripts in spermatozoal populations with the aim for a potential application in fertility prognosis.

• DFG Programme: Research Grants