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