Evaluation of the effect of mitoquinone on functional parameters, DNA structure, and genes expression related to the apoptotic and antioxidants of human sperm after freezing-thawing.

OBJECTIVE: Sperm freezing is considered as an effective way in assisted reproductive technology (ART) programs, it has detrimental effects on sperm function, due to the production of reactive oxygen species (ROS). This study aimed to investigate the potential of Mitoquinone (MitoQ) in inhibiting the production of mitochondrial ROS during sperm freezing. METHODS: A total of 20 human normozoosperm samples were collected for this study. The samples were divided into four groups, each containing different concentrations of MitoQ (0, 0.2, 2, and 20 nM), and then subjected to the freezing process. After thawing, the sperm suspensions were evaluated for parameters including motility, morphology, acrosome integrity, adenosine triphosphate (ATP) level, intracellular ROS, viability, chromatin packaging, DNA denaturation, DNA fragmentation, as well as the expression of antioxidants (GPX, SOD) and apoptotic (Bax, Bcl2) genes. RESULTS: The results showed that total and progressive mobility of sperms significantly increased in the 2 nM group, while significantly decreased in the 20 nM group (p ≤ 0.05). Sperm morphology did not significantly improve across all the tested concentrations (p ≥ 0.05). Intracellular ROS levels showed a significant decrease and increase in the concentrations of 2 and 20 nM, respectively (p ≤ 0.05). Furthermore, a significant increase was observed in viability, ATP, acrosome integrity, chromatin packaging, and non-denatured and non-fragmented DNA after treatment with 2 nM of MitoQ, compared with the control group (p ≤ 0.05). Regarding gene expressions, the relative expressions of oxidative stress genes were increased in the 2 nM group and decreased in the 20 nM group (p ≤ 0.05), while no significant difference was observed in the expressions of apoptotic genes compared with the control group (p ≥ 0.05). All the comparisons were made with respect to the control group. CONCLUSION: Adding the optimal concentration of MitoQ (2 nM) to the sperm freezing medium not only improves sperm functional parameters and reduces DNA damages, but also stimulates the expression of antioxidant genes, leading to even greater benefits for sperm cryopreservation.

Human sperm parameter improvement associated with Ceratonia siliqua extract as a cryopreservation supplement after vitrification.

OBJECTIVE: Given the destructive effects of oxidative stress on sperm structure, this study was conducted to investigate the antioxidant effects of different concentrations of Ceratonia siliqua plant extract on human sperm parameters after the freezing-thawing process. METHODS: A total of 20 normozoospermic samples were frozen. Each sample was divided into two control groups (fresh and cryopreservation) and three cryopreservation experimental groups (containing C. siliqua extract at concentrations of 20, 30, and 40 µg/mL in the freezing extender). Motility, intracellular levels of reactive oxygen species (ROS), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), viability, and acrosome reaction parameters were evaluated. RESULTS: Statistical analysis showed that the highest motility, viability, and PMI were associated with the 20 µg/mL concentration of C. siliqua extract. At all concentrations, intracellular ROS levels were significantly lower and the levels of MMP and the acrosome reaction were significantly higher than in the cryopreservation control group (p≤0.05). CONCLUSION: C. siliqua extract supplements at concentrations of 20, 30, and 40 µg/mL improved sperm motility, viability, PMI, MMP, intracellular ROS, and the acrosome reaction.

Melatonin and selenium supplementation in extenders improves the post-thaw quality parameters of rat sperm.

OBJECTIVE: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. METHODS: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. RESULTS: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). CONCLUSION: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.