Melatonin and Caffeine Supplementation Used, Respectively, as Protective and Stimulating Agents in the Cryopreservation of Human Sperm Improves Survival, Viability, and Motility after Thawing compared to Traditional TEST-Yolk Buffer

Abstract

Cryopreservation processes can damage spermatozoa and impair structural and functional cell characteristics. Plasma, nuclear membranes, and cellular organelles can suffer from the freeze and thaw process. This study evaluates the protective and stimulant effect of melatonin and caffeine supplementation on the functional characteristics of human spermatozoa before and after freezing. Thirty seminal samples from normozoospermic men aged 19-45 years old collected between October 2012 and May 2017 were included. Semen samples were supplemented with either 2 mM melatonin (MEL) prior to cryopreservation, 2 mM caffeine (CAF) in postthaw, or CAF and MEL (CM) in precryopreservation and postthaw, respectively. Kinetics and seminal parameters, mitochondrial activity, DNA fragmentation, and reactive oxygen species (ROS) levels were analyzed before and after cryopreservation. A significant reduction in sperm concentration, total and progressive motility, sperm kinetics, and mitochondrial activity, as well as a significant increase in DNA fragmentation and ROS production in postthaw samples compared to fresh samples, was identified. After administration of a caffeine and/or melatonin supplement, there was a significant increase in progressive motility in the CAF (p = 0.005) and CM (p = 0.048) groups, as well as mitochondrial activity in the CM group (p < 0.05). Cryopreservation has negative effects on overall sperm quality and increases ROS production. A combination of caffeine and melatonin in prefreeze and postthaw sperm samples has proven to be a very effective and simple way to improve semen quality. This will be particularly useful for initial low-quality semen samples, those which suffer the most from the freezing/thawing process.

Figure 1

Sperm structure and energy production mechanisms in the sperm cytoplasm and mitochondria. Energy production is initiated by the glycolytic pathway generating two ATPs and one pyruvate molecule. Pyruvate is essential for the initiation of the acid citric cycle and the oxidative phosphorylation steps of the mitochondrial pathway to produce ATP. Caffeine binds to the adenosine receptor to stimulate the adenylyl cyclase, an enzyme present in mitochondria, leading to the conversion of ATP into cAMP. Protein kinase A (PKA) is activated by cAMP which increases cytochrome c oxidase (COX) activity and oxidative phosphorylation. Caffeine also acts as an inhibitor of phosphodiesterase enzyme activity, inhibiting the degradation of cAMP and promoting an increase in its intracellular concentration.

Figure 2

Study design. CONT group: control (no supplementation) group; CAF group: caffeine treatment group; MEL group: melatonin treatment group; MC group: melatonin+caffeine treatment group.