Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.

Background: Additive manufacturing provides a rapid and flexible alternative to conventional micromolding for producing microneedle systems. This study evaluates the potential of a cost-effective LCD 3D printer for fabricating microneedle arrays (MNAs) and investigates how the geometry of MNAs and the formulation of hydrogel influence the performance of lidocaine-coated arrays. Methods: Conical and pyramidal MNAs, along with a reservoir plate, were designed and manufactured. Lidocaine-loaded and placebo hydrogels with two different polymer concentrations were prepared for dip-coating using both single and multilayer applications. Mechanical resistance and insertion efficiency were evaluated under controlled compression. The physicochemical behavior of the hydrogels were characterized, including pH, spreadability, adhesiveness, and rheological behavior. The uniformity of the coating was analyzed using 3D confocal microscopy. Drug loading was quantified by HPLC, drug release was studied using Franz diffusion cells, and skin penetration was confirmed by 3D confocal imaging and Raman mapping. Results: Conical microneedles exhibited high mechanical integrity, showing only a 2% reduction in height compared to 4% for pyramidal MNAs. Stronger drug signals were achieved in deeper skin layers with the conical geometry, indicating enhanced penetration, while pyramidal MNAs provided slightly higher lidocaine loading due to their larger lateral surface. Hydrogels with higher polymer content produced more stable, uniform coatings, particularly when applied in three layers. Rapid drug release was observed, with over 70% of the drug delivered within minutes. Conclusions: LCD 3D printing offers a cost-effective approach for fabricating MNAs with suitable structural stability and sharpness. The optimized hydrogel formulation ensured uniform coverage, as well as maximal and consistence penetration, making this platform a promising candidate for the dermal delivery of other potent drugs.