The relationship between male genital tract infection, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa

Magister Scientiae (Medical Bioscience) - MSc(MBS)Aim: Leukocytes are the major source of reactive oxygen species (ROS) in the ejaculate and contribute to up to 30% of male infertility. ROS have been associated with markers of apoptosis such as sperm DNA damage, externalization of phosphatidylserine and caspase-3 activation. Therefore, this study aimed at investigating the impact male genital tract infections/inflammations on the induction of apoptosis in spermatozoa.Materials and Methods: Semen samples were obtained from 60 men consulting for fertility problems at the Reproductive Biology Unit, University of Stellenbosch at Tygerberg Academic Hospital, and Vincent Pallotti Hospital (Cape Town, South Africa). To investigate the relationship between male genital tract infection and sperm apoptosis, the following were measured: semen parameters including sperm count, motility and forward progression; oxidative status in the ejaculate by evaluating the concentration of seminal leukocytes, ROS production in the ejaculate,generation of O2-• and H2O2 by spermatozoa, and the activity of reduced glutathione(GSH) in sperm; sperm apoptotic markers by measuring mitochondrial membrane potential (Δψm), caspase-3/7 activation, and DNA fragmentation (TUNEL).Results: The concentration of seminal leukocytes had a significant positive correlation with ROS production in the ejaculate (ρ=0.378; P=0.0064), sperm O2-• production (ρ=0.336; P=0.0098), and caspase-3/7 activation in sperm (ρ=0.527;P<0.0001). Furthermore, at the cutoff value of ≥0.25×106 leukocytes/mL of semen,the concentration of peroxidase-positive cells correlated significantly with sperm GSH activity (ρ=0.718; P=0.008), the percentage of sperm with disrupted Δψm(ρ=0.465; P=0.043), caspase-3/7 activation in sperm (ρ=0.794; P=0.001), and the percentage of sperm with fragmented DNA (ρ=0.563; P=0.017). ROS production in the ejaculate, besides the association with seminal leukocytes, was also correlated with the sperm count (ρ= -0.296; P=0.033), sperm GSH activity (ρ=0.577; P<0.0001),caspase-3/7 activation in sperm (ρ=0.487; P=0.0005), and sperm DNA fragmentation(ρ=0.331 P=0.0171). Caspase-3/7 activation was strongly correlated with oxidative stress in both, the ejaculate and in spermatozoa; although this parameter was not correlated with sperm Δψm and DNA fragmentation. Sperm O2-•, which had a link with seminal leukocyte concentration, was significantly correlated to sperm Δψm(P=0.0098), as was sperm GSH activity (P=0.0055). Sperm DNA fragmentation was positively correlated with ROS in the ejaculate and sperm H2O2-production(P=0.039). Conclusions: Excessive ROS in the ejaculate, mainly a consequence of seminal leukocytes, is not only linked to internal generation of O2-•, but also to sperm DNA fragmentation and the activation of effector caspases. Moreover, even in nonleukocytospermic patients with ≥0.25×106 leukocytes/mL of semen, oxidative stresscan occur which can trigger apoptosis, caspase-3/7 activation, and induce sperm DNA fragmentation. Therefore, it is possible that male genital tract infection, the major cause of leukocyte infiltration in the male reproductive tract, can induce apoptosis, of which the observed sperm DNA fragmentation is a late feature

The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa

The aim of this study was to investigate the relationship between seminal leukocytes, reactive oxygen species (ROS) production in the ejaculate, and markers of apoptosis in human spermatozoa. Semen samples were collected from 60 patients attending fertility clinics at the Reproductive Biology Unit at Tygerberg Academic Hospital and Vincent Pallotti Hospital, Cape Town, South Africa. The concentration of seminal leukocytes was determined and was correlated with ROS production in the ejaculate, the percentage of superoxide (·O2 )- and hydrogen peroxide (H2O2)-positive spermatozoa, glutathione activation in the ejaculate, and with markers of apoptosis in spermatozoa, namely cysteine-dependent aspartate-directed proteases (caspase)-3/7 activation, mitochondrial membrane potential (ΔΨm), and the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive sperm. Significant correlations with the concentration of seminal leukocytes were found for ROS production in the ejaculate, the percentage of ·O2 -positive spermatozoa, and caspase-3/7 activation in the ejaculate. Leukocytospermic samples showed significantly higher ROS production, percentage of ·O2 -positive sperm, GSH activation, and caspase-3/7 activation compared to non-leukocytospermic samples. The percentage of ·O2 -positive sperm was significantly correlated with sperm ΔΨm and caspase-3/7 activation in the ejaculate. Sperm ΔΨm and TUNEL-positive sperm did not correlate with seminal leukocyte concentration. Data demonstrate that high seminal leukocyte concentrations that leads to increased seminal ROS production, and is also associated with caspase activation in the male germ cell and increased mitochondrial ROS production. The latter could possibly be a result of disturbed ΔΨm. The activation of caspase-3/7 could then follow the increased intrinsic superoxide levels due to depleted intrinsic glutathione (GSH). These cellular events might not directly and immediately lead to DNA fragmentation as an endpoint of apoptosis because of topological hindrances.Web of Scienc