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

Abstract

BackgroundSeveral 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.ResultsHere, 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0551-7) contains supplementary material, which is available to authorized users.

Genome-wide discovery and validation of diagnostic DNA methylation-based biomarkers for hepatocellular cancer detection in circulating cell free DNA

“…Use of a broader genome-wide approach as the output for DNA methylation biomarkers in the surrogate tissue of interest, rather than a small marker panel, is an exciting prospect and motivates two interesting future directions to query: 1) could genome-wide assays using small quantities of plasma be used diagnostically, and 2) do these CpGs (hundreds or thousands in this case) perform equally well or better than smaller 5-10 CpG marker panels? Recent research suggests that Illumina Infinium methylation arrays (both the HumanMethylation450k array and the newer generation HumanMethylationEPIC array, which profiles roughly twice the number of sites as the 450k) show promise as a test platform based on interpretation of Clinical Laboratory Improvement Amendments (CLIA) guidelines 57. This could be further strengthened if machine-learning techniques are designed to incorporate all/many probes on the array to make a single call for cases and controls.…”

Sperm DNA Methylation Epimutation Biomarkers for Male Infertility and FSH Therapeutic Responsiveness

“…The current study was designed to identify a molecular biomarker or diagnostic for male idiopathic infertility and provide the proof of concept that an epigenetic analysis will be useful. Previously, the laboratory of Dr. Doug Carrell has utilized an analysis for DNA methylation using a microarray of CpG islands and methylation sites constituting a couple percent of the human genome to identify altered methylation in sperm from infertility patients 15–17 . These previous observations support the potential use of an epigenetic diagnostic for male infertility patients.…”

“…Human studies have also demonstrated a decreased fecundity associated with sperm DNA methylation alterations 13,14 . A sperm DNA methylation biomarker assay has been developed and validated, which uses a microarray approach to assess CpG islands within the genome 15 . Although this analysis only investigates approximately 1% of the genome, it has been shown to be useful in analysis of sperm DNA methylation in a clinical setting 15 .…”

“…A sperm DNA methylation biomarker assay has been developed and validated, which uses a microarray approach to assess CpG islands within the genome 15 . Although this analysis only investigates approximately 1% of the genome, it has been shown to be useful in analysis of sperm DNA methylation in a clinical setting 15 . Subsequently, studies with in vitro fertilization (IVF) applications have used measurement of DNA methylation with this biomarker analysis to assess male infertility prior to assisted reproduction 16–19 .…”

Predicting male fertility from the sperm methylome: application to 120 bulls with hundreds of artificial insemination records

“…Furthermore, from a technical point of view, the starting point for all DNA methylation assays is the preparation of high quality genomic DNA, which is relatively straightforward when compared to other types of epigenetic assays. Standardized platforms such as the Illumina EPIC assay or earlier versions have thus been developed in humans, opening the way to the analysis of large cohorts at a genome scale [ 22 ].…”