Dataset Information

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation.

ABSTRACT:

Background

Several independent research groups have shown that alterations in human sperm methylation profiles correlate with decreased fecundity and an increased risk of poor embryo development. Moving these initial findings from the lab into a clinical setting where they can be used to measure male infertility though requires a platform that is stable and robust against batch effects that can occur between sample runs. Operating parameters must be established, performance characteristics determined, and guidelines set to ensure repeatability and accuracy. The standard for technical validation of a lab developed test (LDT) in the USA comes from the Clinical Laboratory Improvement Amendments (CLIA). However, CLIA was introduced in 1988, before the advent of genome-wide profiling and associated computational analysis. This, coupled with its intentionally general nature, makes its interpretation for epigenetic assays non-trivial.

Results

Here, we present an interpretation of the CLIA technical validation requirements for profiling DNA methylation and calling aberrant methylation using the Illumina Infinium platform (e.g., the 450HM and MethylationEPIC). We describe an experimental design to meet these requirements, the experimental results obtained, and the operating parameters established.

Conclusions

The CLIA guidelines, although not intended for high-throughput assays, can be interpreted in a way that is consistent with modern epigenetic assays. Based on such an interoperation, Illumina's Infinium platform is quite amenable to usage in a clinical setting for diagnostic work.

SUBMITTER: Abbasi M

PROVIDER: S-EPMC6145208 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Publications

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation.

Abbasi Mohammad M Smith Andrew D AD Swaminathan Harish H Sangngern Peer P Douglas Amanda A Horsager Alan A Carrell Douglas T DT Uren Philip J PJ

Clinical epigenetics 20180918 1

<h4>Background</h4>Several independent research groups have shown that alterations in human sperm methylation profiles correlate with decreased fecundity and an increased risk of poor embryo development. Moving these initial findings from the lab into a clinical setting where they can be used to measure male infertility though requires a platform that is stable and robust against batch effects that can occur between sample runs. Operating parameters must be established, performance characteristi ...[more]

PMID: 30227883

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Project description:DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A) transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation changes induced by hDNMT3A expression were intergenerationally inherited by offspring without transmission of the transgene, which provided evidence for the transmission of active endogenous-factors-induced epigenetic variations.

Project description:BACKGROUND: In previous studies using candidate gene approaches, low sperm count (oligospermia) has been associated with altered sperm mRNA content and DNA methylation in both imprinted and non-imprinted genes. We performed a genome-wide analysis of sperm DNA methylation and mRNA content to test for associations with sperm function. METHODS AND RESULTS: Sperm DNA and mRNA were isolated from 21 men with a range of semen parameters presenting to a tertiary male reproductive health clinic. DNA methylation was measured with the Illumina Infinium array at 27,000 CpG loci. Unsupervised clustering of methylation data differentiated the 21 sperm samples by their motility values. Recursively partitioned mixture modeling (RPMM) of methylation data resulted in four distinct methylation profiles that were significantly associated with sperm motility (P=0.01). Linear models of microarray analysis (LIMMA) was performed based on motility and identified 9,189 CpG loci with significantly altered methylation (Q<0.05) in the low motility samples, with many loci located in genes associated with subfertility and epigenetic regulation. In the low motility samples, the majority of disrupted CpG loci (80%) were hypomethylated. Of the aberrantly methylated CpGs, 194 were associated with imprinted genes almost equally distributed into hypermethylated (predominantly paternally expressed) and hypomethylated (predominantly maternally expressed) groups. Sperm mRNA was measured with the Human Gene 1.0 ST Affymetrix GeneChip Array. LIMMA analysis based on motility identified 20 candidate transcripts as differentially expressed in low motility sperm, including HDAC1 (NCBI 3065), SIRT3 (NCBI 23410), and DNMT3A (NCBI 1788). Altered expression of these epigenetic regulatory genes was associated with RPMM DNA methylation class. CONCLUSIONS: Using integrative genome-wide approaches to study epigenetic and gene expression patterns in human sperm we identified CpG methylation profiles and mRNA alterations associated with low sperm motility, and that low motility sperm may have aberrant genome-wide hypomethylation due to excess HDAC1 activity. See summary above

Dataset Information

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation.

Background

Results

Conclusions

Publications

Establishing a stable, repeatable platform for measuring changes in sperm DNA methylation.

Similar Datasets

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