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

特发性不育男性高度纯化的精子和精浆中的 mRNA 和小非编码 RNA

• 批准号: 214733733
• 负责人: Dr. Andrej-Nikolai Spiess
• 金额: --
• 依托单位: Zentrum für Innere Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/214733733?language=en
• 关键词: mRNAs; small; non; coding; RNAs

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.

在辅助生殖技术（ART）的生育结果的预测是不可靠的，当仅仅基于精子图参数。因此，近年来进行了几项研究，其中人类射精的基因表达模式与ART结果相关。在我们资助的项目中，我们采用体外受精程序的射精来研究不同结果（妊娠，未妊娠，卵母细胞未受精）的差异基因表达。为了提高统计能力，我们使用了一种特殊的数据聚合技术，包括和重新分析四个已发表的基因表达数据集。随后筛选的差异基因适合验证。我们的重新分析表明，所有已发表的ART结果预后的基因表达数据集都被体细胞转录本大量污染，我们通过在基因表达分析之前不充分消除“圆形细胞”（白细胞，巨噬细胞，上皮细胞）来解释这一观察结果。将通过荧光原位杂交研究精子上或精子中体细胞转录本的假定生育调节结合/掺入。将对表现为正常精子症的可育和特发性不育男性进行基于RNA测序的分析，以揭示伴随正常精子图参数的特发性不育中的差异表达。初步结果表明特异性和高表达的piRNA对精子功能的潜在作用。此外，我们还将对相应精浆的小RNA进行测序，以揭示细胞和精浆组分之间可能存在的转录本转移。很明显，精子群体的基因表达异质性很强，可能导致几乎不可能鉴定出与生育力相关的转录本。一个潜在的补救措施，这个问题可能会发现在基因表达分析的单精子在高通量的方式，以揭示一个假定的异质性分布的生育力相关的转录在精子人口。在与微系统技术研究所（IMTEK，弗赖堡）的合作中，我们优化了他们开发的单细胞打印机的协议，以实现在微量滴定板中高通量沉积单个精子（“精子打印”）。对已知的或从我们的差异分析获得的转录本进行下游定量实时PCR将作为原理证明，以揭示转录本在精子群体中的异质性分布，目的是在生育力预后中的潜在应用。