The Effect of Non-Thermal Plasma on the Structural and Functional Characteristics of Human Spermatozoa

Abstract

Significant antibacterial properties of non-thermal plasma (NTP) have converted this technology into a promising alternative to the widespread use of antibiotics in assisted reproduction. As substantial data available on the specific in vitro effects of NTP on male reproductive cells are currently missing, this study was designed to investigate selected quality parameters of human spermatozoa (n = 51) exposed to diffuse coplanar surface barrier discharge NTP for 0 s, 15 s, 30 s, 60 s and 90 s. Sperm motility characteristics, membrane integrity, mitochondrial activity, production of reactive oxygen species (ROS), DNA fragmentation and lipid peroxidation (LPO) were investigated immediately following exposure to NTP and 2 h post-NTP treatment. Exposure to NTP with a power input of 40 W for 15 s or 30 s was found to have no negative effects on the sperm structure or function. However, a prolonged NTP treatment impaired all the sperm quality markers in a time- and dose-dependent manner. The most likely mechanism of action of high NTP doses may be connected to ROS overproduction, leading to plasma membrane destabilization, LPO, mitochondrial failure and a subsequent loss of motility as well as DNA integrity. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully selected in order to preserve the sperm vitality, should NTP be used in the practical management of bacteriospermia in the future.

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men

“…Subsequently, 150 μL of the diluted semen sample was mixed with 50 of the IDENT stain (final concentration of 10 μg/mL; Hamilton-Thorne Biosciences, Beverly, MA, USA) and incubated at 37 °C for 10 min. Then, 10 μL of the mixture was placed into the analyzer and at least 300 cells were assessed under fluorescent illumination [ 64 , 65 ].…”

“…Commercially available Annexin-V-FLUOS kit (Roche Applied Science, Basel, Switzerland) was used to evaluate the sperm membrane stability. A dual staining protocol with Annexin-V (AV) and propidium iodide (PI) enabled to distinguish cells in the early stage of apoptosis (AV + /PI 2212 ), necrotic or late-stage apoptotic cells (AV + /PI + ) or living cells (AV 2212 /PI 2212 ) [ 65 ]. Sperm suspensions adjusted with pre-warmed PBS (Sigma-Aldrich, St. Louis, MO, USA) to 10 6 cells were pre-treated with 50 μL of incubation buffer, 1 μL AV and 1 μL PI.…”

“…Sperm suspensions adjusted with pre-warmed PBS (Sigma-Aldrich, St. Louis, MO, USA) to 10 6 cells were pre-treated with 50 μL of incubation buffer, 1 μL AV and 1 μL PI. Following incubation at 37 °C for 30 min, the samples were stained with 10 μL DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich, St. Louis, MO, USA; 1 μM in PBS), and at least 300 cells were observed under an epifluorescence microscope with a 40× magnification objective (Leica Microsystems, Wetzlar, Germany) [ 65 , 66 ]. The data are expressed as % apoptotic cells and % necrotic cells, respectively.…”

“…Following incubation at 37 °C for 20 min, the cells were centrifuged at 150× g and 20 °C for 10 min, washed twice with PBS, transferred to a black 96-well plate and JC-1 monomers and polymers were quantified with a combined spectro-fluoro-luminometer (Glomax Multi+; Promega Corporation, Madison, WI, USA). Mitochondrial membrane potential (ΔΨm) is expressed as the ratio of JC-1 complexes to JC-1 monomers (red/green ratio) [ 65 ].…”

“…The light signal emitted from the interaction of ROS and luminol was monitored with the Glomax Multi + combined spectro-fluoro-luminometer (Promega Corporation, Madison, WI, USA). The experimental results are expressed in relative light units per second per million sperm (RLU/s/10 6 sperm) [ 65 ].…”