Assessment of sperm DNA fragmentation and methylation for prediction of male fertility

Abstract

Infertility presents a significant global health challenge affecting around one-sixth of the population, with 30-50% of cases attributed to male infertility. Conventional semen analysis has been relied upon the World Health Organization (WHO) guidelines to assess male fertility based on parameters such as motility and morphology. However, its limited predictive power for sperm fertilising capacity and fertilisation outcome remains controversial. Sperm DNA fragmentation (sDF) has been shown to correlate with suboptimal artificial reproductive treatment (ART) outcomes such as lower blastulation rate or defective embryonic development. The incorporation of a standardised test for sperm DNA evaluation can improve the predictive power in assessing male infertility as it provides information from the genetic level.

Sperm DNA methylation is another proposed method in identifying infertile patients, and prior investigations have shown that DNA methylation is correlated to sperm function and quality. Methylation is the most prominent mechanism in sperm epigenetic regulation; chromatin modification and post-transcriptional regulation are very limited in spermatozoa due to the minimal amount of cytoplasm and RNA presented in the spermatozoa. Another factor is that the sperm genome is inherently transcriptional and translational silence. Our study aimed to identify biomarkers related to sperm DNA fragmentation and to understand the association between sperm DNA fragmentation and methylation.

In our retrospective study involving 135 infertile patients undergoing in-vitro fertilisation treatment, the TUNEL assay along with flow cytometry was employed as the standardised method for sDF assessment. Our findings revealed that elevated sDF rates were associated with advanced male age (p<0.001), with males over 38 years exhibiting significantly higher sDF levels compared to younger individuals (p<0.01). Moreover, sDF rates were negatively linked to normal fertilisation rates in male infertile couples (p<0.05) and embryo utilisation rates (p<0.01).

Regarding the association between sDF and methylation markers, samples were isolated from raw semen, swim-up samples, those with 0% normal WHO morphology, and normal morphology samples in our study. The global methylation levels across these samples remained consistent. Enzymatic methylation sequencing analysis comparing among sample groups highlighted gene ontology pathways related to cellular respiration and mitochondrial function. Notably, MT-ND2 emerged as a potential methylation biomarker linked to reactive oxygen species (ROS) production impacting sperm DNA integrity. MT-ND2 variants in males have been associated with reduced fertility, underscoring its significant role in male reproductive health in conjunction with our study's findings.