Effect of sperm cryopreservation and preparation before freezing–thawing process: A study of DNA fragmentation and ultrastructure

A BSTRACT Male fertility preservation can be achieved through assisted reproductive technology, in which sperm cryopreservation plays an essential role. However, the freezing–thawing process induces cellular stress, primarily through intracellular ice crystal formation, which damages membranes, organelles, and exacerbates osmotic and oxidative stress. This study aims to determine whether sperm cryopreservation and preparation affect sperm concentration and motility, cryosurvival rate (CSR), DNA fragmentation, and ultrastructure after thawing. A total of 10 human sperm samples were rapidly frozen in cryovials, with prefreeze and postthaw analyses compared against fresh controls, assessing sperm parameters, DNA fragmentation, and ultrastructure (scanning electron microscope). Statistical analysis included paired comparisons for two groups and multigroup tests with post hoc analysis. CSR was compared to 100% using a one-sample t -test. P < 0.05 was considered statistically significant. Sperm concentration decreased both after washing and after thawing compared to before freezing, while motility increased after washing, but it significantly decreased after thawing. The sperm motility result was confirmed by a significantly reduced CSR and DNA fragmentation index, which significantly increased at after thawing. Ultrastructure analysis also revealed more sperm damage after thawing. Cryopreservation impairs sperm motility, CSR, DNA fragmentation, and ultrastructure. Since in vitro fertilization requires sperm with good morphology and motility, new strategies are needed to minimize cryodamage.