Expression analysis of small noncoding RNA (sRNA), including microRNA, piwi-interacting RNA, small rRNA-derived RNA, and tRNA-derived small RNA, is a novel and quickly developing field. Despite a range of proposed approaches, selecting and adapting a particular pipeline for transcriptomic analysis of sRNA remains a challenge. This paper focuses on the identification of the optimal pipeline configurations for each step of human sRNA analysis, including reads trimming, filtering, mapping, transcript abundance quantification and differential expression analysis. Based on our study, we suggest the following parameters for analysis of human sRNA in relation to categorical analyses with two groups of biosamples: (1) trimming with the lower length bound = 15 and the upper length bound = \(Read\ length - 40\% Adapter\ length\); (2) mapping on a reference genome with bowtie aligner with one mismatch allowed (-v 1 parameter); (3) filtering by mean threshold > 5; and (4) analyzing differential expression with DESeq2 with adjusted p-value < 0.05 or limma with p-value < 0.05 if there is very little signal and few transcripts.

Advanced paternal age at fertilization has been suggested to be a risk factor for neurodevelopmental, psychiatric and other disorders in offspring. One emerging hypothesis suggests that altered offspring phenotype is linked with age-related accumulation of epigenetic changes in the sperm of fathers. Given that paternal age is increasing in the developed world, understanding aging-related epigenetic changes in sperm is needed as well as environmental factors that modify such changes. In this study, we characterize age-dependent changes in sperm DNA methylation profiles between young pubertal (postnatal day (PNDs) 65) and mature (PND120) Wistar rats. We also analyze these changes in rats exposed perinatally to 0.2 mg/kg of ubiquitous environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). Reduced representation bisulfite sequencing (RRBS) libraries were prepared from caudal epididymal sperm DNA and differentially methylated regions (DMRs; ≥ 10x coverage depth, ≥ 3 CpGs per cluster, ≥ 5% methylation change, q < 0.05) were identified via MethPipe package. In control animals, 5,319 age-dependent DMRs were identified, with 99.3% DMRs hypermethylated in mature animals compared to young pubertal rats. These age-related DMRs were enriched for functional categories essential for embryonic development, such as pattern specification, forebrain and sensory organ development, Hippo and Wnt pathways. Age-related changes in sncRNA, reported in different study, target similar list of genes and biological categories.In BDE-47 exposed rats, sperm DNA methylation was higher in young pubertal and lower in mature animals when compared to controls, which resulted in a significant attenuation in the number of age-dependent DMRs (N = 189) identified in the exposed group. In conclusion, our results indicate that the natural aging process has profound effects on sperm methylation levels and this effect may be modified by environmental exposures. Moreover, our results further support the role of epigenetic mechanisms as a likely link betwen paternal age and offspring health and development.

Advanced paternal age at fertilization is a risk factor for multiple disorders in offspring and may be linked with age-related epigenetic changes in fathers sperm. Understanding of aging-related epigenetic changes in sperm and environmental factors that modify such changes is needed. Here we characterize changes in sperm sncRNA between young pubertal and mature rats. We also analyze modification of these changes by exposure to environmental xenobiotic 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47). SncRNA libraries prepared from epididymal spermatozoa were sequenced and analyzed using DESeq 2. Distribution of small RNA fractions changed with age, with fractions mapping to rRNA and lncRNA decreasing and fractions mapping to tRNA and miRNA increasing. 249 miRNA, 908 piRNA and 227 tRNA-derived RNA were differentially expressed (2-fold change, FDR p ≤ 0.05) between age groups in control animals. Differentially expressed miRNA and piRNA were enriched for protein-coding targets involved in development and metabolism, piRNA were enriched for LTR targets. BDE-47 accelerated age dependent changes in sncRNA in younger animals, decelerated these changes in older animals and increased the variance in expression of all sncRNA. Our results indicate that the natural aging process has profound effects on sperm sncRNA profiles and this effect may be modified by environmental exposures.