Determination of leucocyte extracellular traps (ETs) in seminal fluid (ex vivo ) in infertile patients—A pilot study

Abstract

Leucocytospermia has been associated with loss of sperm function. Extracellular traps (ETs) of leucocytes are produced during innate immune response. ETs can be activated by spermatozoa in contact with polymorphonuclear (in vitro), inducing sperm entrapment and decrease motility. In this pilot study, we describe the results of ETosis ex vivo, in seminal fluid (SF) smear of infertile patients, associating ETs with leucocytospermia and bacteriospermia. In 21 infertile patients, semen parameters (WHO, 2010), microbiological study, leucocytospermia and presence of ETs in SF were determined. Leucocytes (CD45, CD15 and CD68) were evaluated by immunostaining in SF smears. Indirect immunofluorescence (global histone and H4‐citrullinated 3) and scanning electron microscopy (SEM) were used to determine ETs morphology. In 28.6% of patients presented leucocytospermia without bacteriospermia, all of them presented a large number of ETs in the SF smears examined. About 76.6% of the patients without leucocytospermia were positive for ETs. Samples with leucocytospermia have a higher number of ETs and would be related to the amount of leucocytes in the SF. The morphological predominant ETs were diffuse (diffETs) and spread (sprETs). The formation of ETs indicates leucocyte activation in semen, and it was observed that ETosis does not depend exclusively on the presence of bacterial contamination.

Increase of leucocyte-derived extracellular traps (ETs) in semen samples from human acute epididymitis patients—a pilot study

“…Sperm-mediated ET formation by seminal neutrophils and macrophages in vitro and ex vivo was recently reported to occur in different mammal species including humans [12,21,22,30,31]. Here we investigated for the first time the possible interactions of human neutrophils/macrophages in semen samples obtained from acute epididymitis patients, New evidence indicates adverse effects of neutrophils and/ or macrophages on sperm function after exposure to extruded ETs [12,21,22]. Although it is known that seminal fluid contains macrophages and neutrophils as well as spermatozoa [32].…”

“…So far, neutrophils can cast all three characteristic morphologies, whilst macrophages/monocytes mainly release diffETs [22]. These morphological differences are often related with the type of stimulus, causing the formation of different ETs [21,22]. Interestingly, recent studies dealing with infertility in male patients have related extrusion of aggETs with leucocytospermia and bacteriospermia, which would stimulate both migration and a marked response of these urogenital-derived leucocytes [37].…”

“…Interestingly, recent studies dealing with infertility in male patients have related extrusion of aggETs with leucocytospermia and bacteriospermia, which would stimulate both migration and a marked response of these urogenital-derived leucocytes [37]. Conversely, presence of all three types of ETs have also been described in human seminal fluid with no microbial infection; these are most probably associated with other conditions-such as chronic aseptic inflammation or immunological disorderswhich would result in the stimulation of seminal neutrophils and macrophages [12,21].…”

“…ETs are highly effective antimicrobial structures, capable of capturing and eliminating foreign agents such as bacteria, fungi, viruses, and large-sized parasites [15,[18][19][20]. To date, different in vitro, ex vivo, and in vivo studies in the reproduction of domestic animals have been published, showing signalling pathways in detail and highlighting the biological importance of ETosis against sperm and pathogens [21]. However, little is known to date about their role in human reproduction, and much less about their pivotal role in the immunopathogenesis of infectious diseases of the male urogenital tract [21,22].…”

Changes in Haematological Parameters Following Toxicity Study with 80% Methanol Extract of <i>Moringa oleifera</i> in Wistar Rats

“…(viii) Determination of Total Leucocyte (WBC) Count Determination of leucocyte count was carried out as described by [30]. Blood was drawn up to 0.5 marks on the stem of the diluting pipette and held horizontally; the external surface of tip was wiped out clearly using a clean cotton wool.…”