Seminal plasma lipid peroxidation

A statistically significant negative correlation was found between TBARS levels and seminal plasma pH. The preservation of the semen pH within its reference ranges (7.2–8.2) is of great importance for the regulation of various physiological sperm functions (Zhou et al., 2015). This correlation could possibly be explained by the fact that at physiological levels of pH in seminal plasma MDA, the major end-product of lipid peroxidation, is present as an enolate ion with low reactivity. However, lowering

the pH causes the formation of highly reactive compound known as beta-hydroxyacrolein, which can react with other molecules in the vicinity and cause a considerable increase in lipid peroxidation (Papac-Milicevic et al., 2016). Furthermore, the accumulation of MDA in a highly acidic milieu results in the formation of long oligomers, which causes hydrolytic cleavage of recently produced MDA oligomers. This process ends with the formation of additional highly immunogenic epitops on major cellular macromolecules that play a part in secondary deleterious reactions (Ayala et al., 2014;

Papac-Milicevic et al., 2016).

Seminal plasma TBARS levels were significantly and negatively correlated with the proportion of rapid spermatozoa. A similar, though non-significant, trend was observed with regards to the proportions of progressive and total motility, whereas the correlation of TBARS levels with the proportions of medium and slow spermatozoa were significant and positive. The negative correlation between lipid peroxidation and sperm quality parameters of motility has been reported in a number of studies (Akbari et al., 2010; Colagar et al., 2013; Patel et al., 2009). Interestingly, substantially elevated concentrations of MDA were found in sperm pellet suspensions (Suleiman et al., 1996;

Tavilani et al., 2005) and seminal plasma (Colagar et al., 2013) of patients with asthenospermic compared to normozoospermic fertile men. In addition, the in vitro exposure of human spermatozoa to electrophilic lipid aldehydes, such as acrolein and 4-hydroxynonenal (4HNE) produced by lipid peroxidation, resulted in a significant decline in both total and progressive motility percentage, while sperm viability was not compromised (Aitken et al., 2012). The findings of the current study also show that the detrimental effect of lipid peroxidation on sperm motility could occur even before cell death was observed.

This study also found a statistically significant negative correlation between TBARS levels and the sperm kinematics VCL, VSL, VAP and BCF. Similar but non-significant trends were observed with regards to percentage of ALH. The parameters VCL, VSL and VAP are measures of sperm progressive velocity and are revealed to play a vital role in sperm competition (Malo et al., 2005).

They have also been suggested as potential reliable indicators of male fertility (Farooq et al., 2017;

Nagy et al., 2015; Santolaria et al., 2015). BCF is one of the useful parameters that contribute substantially to the overall sperm linear progression. It indicates the rate at which the curvilinear path crosses the average path; however, it may vary in value depending on the VAP setting on the CASA instrument (King et al., 2000; Lu et al., 2014). The sensitivity of these parameters to the deleterious effects of lipid peroxidation appears to be higher than that of the percentage motility, which was not correlated with TBARS levels in this study.

Although the concentration of seminal plasma TBARS was not correlated with sperm viability, significantly elevated levels were observed in samples with sperm viability percentages below that of the WHO reference values compared to those with normal values (p<0.05). The optimal cut-off value to distinguish between samples with compromised and normal viability on the basis of TBARS was 9.855 µmol/L. At this cut-off value, the positive and negative predictive values of the test were 97 % and 36 % respectively. The distribution of TBARS levels for the two groups above and below the WHO viability reference value as well as sensitivity and specificity of the test are given in Figure 4.26 A and B.

In general, the most remarkable effect of lipid peroxidation on cellular function is related to the physicochemical properties of cellular and organelle membranes. The specific lipid composition of these membranes is essential to maintain the overall normal sperm function (Agarwal et al., 2014b;

Ayala et al., 2014). Spermatozoa membranes contain an extraordinary high content of PUFA (Henkel, 2011). The rate of fatty acid oxidation can differ according to their chain length, degree of unsaturation, and position and configuration of double bonds (DeLany et al., 2000). The distribution of the lipid composition of the sperm plasma membrane has been shown to be regionally different, allowing for the distinctive functions of these domains (Connor et al., 1998). As compared to the sperm head membrane, the sperm tail is estimated to contain substantially higher amounts of total and individual unsaturated fatty acids, mainly docosahexaenoic and arachidonic acids (Connor et al., 1998). The enrichment of the tail membrane with these unsaturated fatty acids contributes to its fluidity and flexibility, which is critical for tail motility (Agarwal et al., 2014b; Rooke et al., 2001).

These fatty acids are also known to be amongst the most biologically active (Else and Kraffe, 2015), which further enhances the susceptibility of the sperm tail to oxidative alterations.

The role of mitochondria in energy production for human sperm motility has been established (Piomboni et al., 2012). Dysfunctions of mitochondrial membrane integrity may represent a major feature of sperm motility impairment (Paoli et al., 2011; Pelliccione et al., 2011). The inner mitochondrial membrane is especially important, as it is the site of electron transport chain and ATP synthesis. This membrane is highly susceptible to lipid peroxidation due to its high PUFA content and close proximity to ROS production within the mitochondria (Castilho et al., 1999; Kalogeris et al., 2014). Unlike the plasma membrane, the inner mitochondrial membrane exclusively contains high levels of the phosphatidylglycerol cardiolipin which is particularly prone to peroxidation (Paradies et al., 2009; Schenkel and Bakovic, 2014). Under normal physiological conditions, the inner mitochondrial membrane is selectively permeable to only neutral small molecules. However, in case of oxidative stress, peroxidation of mitochondrial membrane phospholipids can trigger alterations mitochondrial membrane potential (Piomboni et al., 2012; Wei et al., 2001). These changes have recently been suggested to be an early indicator of motility impairment as they occur in parallel to or even prior to any detectable changes in overall sperm motility (Agnihotri et al., 2016; La Piana et al., 1998). This further supports the importance of rapid progressive motility linear velocity variables as early and sensitive indicators of lipid peroxidation that could be impaired prior to any detectable deterioration in other sperm motion characteristics.