Ejaculatory Abstinence Affects the Sperm Quality in Normozoospermic Men-How Does the Seminal Bacteriome Respond?

Abstract

This study was designed to describe bacterial profiles of ejaculates collected following a long and short ejaculatory abstinence set in the context of changes in the conventional, oxidative, and immunological characteristics of semen. Two specimens were collected in succession from normozoospermic men (n = 51) following 2 days and 2 h, respectively. Semen samples were processed and analyzed according to the World Health Organization (WHO) 2021 guidelines. Afterwards, sperm DNA fragmentation, mitochondrial function, levels of reactive oxygen species (ROS), total antioxidant capacity, and oxidative damage to sperm lipids and proteins were evaluated in each specimen. Selected cytokine levels were quantified using the ELISA method. Bacterial identification by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that samples collected following two days of abstinence presented with a higher bacterial load and diversity, and a greater prevalence of potentially uropathogenic bacteria including Escherichia coli, Staphylococcus aureus and Enterococcus faecalis. Only staphylococci and Escherichia coli remained present in specimens obtained after 2 h of abstinence. Whilst all samples accomplished the criteria set by WHO, a significantly higher motility (p < 0.05), membrane integrity (p < 0.05), mitochondrial membrane potential (p < 0.05), and DNA integrity (p < 0.0001) were detected following 2 h of ejaculatory abstinence. On the other hand, significantly higher ROS levels (p < 0.001), protein oxidation (p < 0.001), and lipid peroxidation (p < 0.01) accompanied by significantly higher concentrations of tumor necrosis factor alpha (p < 0.05), interleukin-6 (p < 0.01), and interferon gamma (p < 0.05) were observed in specimens collected after two days of abstinence. It may be summarized that shorter ejaculatory abstinence does not compromise sperm quality in normozoospermic men, while it contributes to a decreased occurrence of bacteria in semen which is accompanied by a lower probability of damage to spermatozoa by ROS or pro-inflammatory cytokines.

Keywords: apoptosis; bacteria; ejaculatory abstinence; inflammation; oxidative stress; semen quality.

Figure 1

Variations in the semen volume (a), sperm concentration (b) and motility (c) following 2 days and 2 h of ejaculatory abstinence. * p < 0.05; *** p < 0.001.

Figure 2

Variations in the membrane integrity (a), acrosome integrity (b), mitochondrial membrane potential (c) and DNA integrity (d) following 2 days and 2 h of ejaculatory abstinence. * p < 0.05; **** p < 0.0001; ns—non-significant.

Figure 3

Variations in the levels of reactive oxygen species (ROS) in neat semen (a), total antioxidant capacity of the seminal plasma (b), oxidative damage to the proteins (c) and lipids (d) assessed in the sperm lysates following 2 days and 2 h of ejaculatory abstinence. ** p < 0.01; *** p < 0.001; ns—non-significant.

Figure 4

Variations in the concentration of leukocytes found in neat semen (a), levels of tumor necrosis factor alpha (b), interleukin-1β (c), interleukin-6 (d), interferon gamma (e) and C-reactive protein (f) in the seminal plasma following 2 days and 2 h of ejaculatory abstinence. * p < 0.05; ** p < 0.01; *** p < 0.001; ns—non-significant.

Figure 5

Expression patterns of BAX (a) and Bcl-2 protein (b) in semen samples collected following 2 days and 2 h of ejaculatory abstinence. ns—non-significant. Original photos of the blots are available as Supplementary Materials (Figure S1: Original blot of BAX protein; Figure S2: Inverted blot of BAX protein; Figure S3: Original blot of Bcl-2 protein; Figure S4: Inverted blot of Bcl-2 protein; Figure S5: Original blot of beta actin; Figure S6: Inverted blot of beta actin). Created with BioRender.com (accessed on 27 November 2022).

Figure 6

Krona charts of the bacteria recovered from semen samples collected following 2 days (a) and 2 h (b) of sexual abstinence (outermost ring: species, middle ring: genus, innermost ring: family).