Background: Perm freezing compromises nuclear integrity. Standardized tools to assess three-dimensional (3D) chromatin alterations are lacking.

Objectives: This study aimed to present a novel protocol for 3D nuclear morphometric analysis of human spermatozoa to measure slow freezing and thawing-induced alterations.

Materials and methods: Human sperm cells were examined before and after freezing using a consistent 3D nuclear morphometric analysis protocol that distinguishes between live and dead spermatozoa. Morphometric assessments were performed by microscopy and image analysis with the NucleusJ2.0/NODeJ software, without introducing denaturing agents. The analysis included measurements of nuclear volume, elongation, flatness, and the volume of hypercondensed chromatin (Hc) regions, along with the relative fluorescence intensity of these zones (RHF intensity). Additional parameters evaluated comprised sperm vitality, motility, DNA fragmentation, and chromatin decondensation.

Results: A quantitative analysis of 4919 sperm nuclei from 10 patients demonstrated significant modifications in the hypercondensed chromatin (Hc) zones, with a marked decrease in sperm vitality and motility (p < 0.001) and a significant increase in DNA fragmentation (p < 0.05). At the chromatin level, slow freezing induced a higher number of Hc zones per nucleus (p < 0.01), a reduction in the average volume of these zones (p < 0.0001), and a reduced relative fluorescence intensity (p < 0.01). Notably, these chromatin alterations were most pronounced in viable spermatozoa.

Discussion and conclusion: This study is the first to provide standardized 3D nuclear morphometry measurements for human sperm, offering a novel biomarker to assess male fertility and cryopreservation susceptibility, with potential clinical applications for improving patient care. Slow freeze-thaw cycle induced significant alterations in sperm chromatin, disrupting nuclear organization and forming several smaller, less compacted hypercondensed chromatin zones.