DNA fragmentation and hydroxymethylation in ejaculated spermatozoa in normozoospermia and pathozoospermia

BACKGROUND: The search for new criteria for semen quality based on the evaluation of the structural and functional state of the sperm genome remains a relevant task in reproductive medicine.

AIM: This work aimed to assess DNA integrity and the content of 5-hydroxymethylcytosine (5hmC) in the same ejaculated spermatozoa obtained from patients with normozoospermia and pathozoospermia.

METHODS: The study included semen samples from 63 patients with normozoospermia (n = 33) and pathozoospermia (n = 30). Microscopic slides were prepared from the samples, on which fragmented DNA was first detected using the TUNEL assay, followed by digital image acquisition. Subsequently, 5hmC was detected by indirect immunofluorescence, and digital images of the same microscopic fields were acquired again. In total, 126,000 spermatozoa were analyzed (2000 per sample).

RESULTS: A substantial proportion of spermatozoa (72.8%–94.2%) in all samples showed no DNA integrity violations and exhibited a low (background) level of 5hmC. The proportions of spermatozoa exhibiting DNA fragmentation, increased hydroxymethylation, or both characteristics simultaneously were 0.05%–13.8%, 0.15%–11.5%, and 0.99%–13.38%, respectively. The proportion of spermatozoa with DNA fragmentation and/or DNA hyperhydroxymethylation did not differ between patients with normozoospermia and those with pathozoospermia. DNA fragmentation and DNA hyperhydroxymethylation in ejaculated spermatozoa were found to be interdependent, and their coexistence in gametes was nonrandom.

CONCLUSION: The nonrandom coexistence of DNA fragmentation and DNA hyperhydroxymethylation in spermatozoa, along with the interdependence of these features, indicates a common trigger, most likely oxidative stress. However, the presence of additional factors leading to DNA damage or altered hydroxymethylation levels may explain the less than complete overlap of these features in spermatozoa. The assessment of DNA fragmentation and hydroxymethylation levels in spermatozoa appears to be a promising approach for evaluating semen quality and identifying potential causes of idiopathic infertility.