Impact of Endocrine Disruptors on Mirna Expression in Sperm: A Risk Factor for Autism?

Background: The rapidly rising prevalence of autism spectrum disorders (ASD) in the U.S., now as high as 1 in 40 individuals, suggests that environmental factors may be increasing risk for ASD. However, there is a critical gap in our knowledge regarding the nature of environmental factors and the biological mechanisms through which they may influence susceptibility to ASD. Recent studies by our group and others suggest that endocrine disrupting compounds (EDCs), many of which are ubiquitous in our environment, may be contributing to the rising prevalence of ASD. Moreover, germline exposures to EDCs in animal models have been reported to induce lasting phenotypic changes in offspring which are, in some cases, transmitted transgenerationally and potentially through epigenetic mechanisms.

Objectives: The overarching goal of this study is to test the hypothesis that miRNA expression in sperm is associated with measured levels of 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p’-DDE) in the serum of young sperm donors from the Faroe Islands, whose diet includes pilot whale meat and blubber which predisposes them to higher than average levels of long-lived environmental EDCs, which accumulate in fatty tissues. Specifically, we investigated differences in the expression of miRNAs in sperm from men with the highest (third tertile) and lowest (first tertile) exposures with respect to blood p,p’-DDE levels, and performed bioinformatics analyses on the differentially expressed miRNAs to identify target genes and pathways that may predispose offspring to ASD.

Methods: Sperm from men with the highest and lowest levels of DDE exposure (8 samples per group) was isolated from whole semen using a density gradient protocol. Total RNA was obtained from cell lysates (after DNA extraction) using Ribozol followed by chloroform extraction. The isolated RNA was submitted for small RNAseq analysis. RNAseq data was analyzed using the MicroRNA Analysis app within Illumina’s BaseSpace Sequence Hub. Ingenuity Pathways Analysis (IPA) software was used to identify the potential target genes of differentially expressed miRNAs, which were then analyzed for pathways and functions using the Core Analysis feature of IPA.

Results: Fifty-five and 132 differentially expressed miRNAs were identified by the DESeq2 and RankProd software packages, respectively, in the MicroRNA Analysis app, with 25 miRNAs represented in both datasets. Approximately 6300 potential target genes were identified for the 25 overlapping miRNAs, of which 347 were represented in SFARI Autism Gene Database (hypergeometric distribution p-value ≤ 0.015 for enrichment in autism risk genes). Network prediction analysis of the 347 overlapping genes revealed GABA receptor signaling (p-val: 5.5E-06) and PTEN signaling (p-val: 1.1E-05) as the top canonical pathways represented within the gene set, with an overabundance of genes involved in developmental disorder (100 genes, p-val: 4.8E-05 – 1.2E-39), neurological disease (211, p-val: 6.2E-05 – 5.4E-35), and nervous system development and function (50, p-val: 1.7E-05 – 1.8E-13). Seventy of these genes are directly linked to autism/intellectual disability (p-val: 1.2E-39).

Conclusions: The results of this pilot study show that higher levels of exposure to p,p’-DDE are associated with differentially expressed microRNAs in sperm. These miRNAs potentially target genes involved in pathways and functions implicated in ASD.