Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility

We investigated the interaction of sperm with neutrophil extracellular traps and determined the major proteins involved and observed that the release is time and dose dependent and that it decreases sperm motility.

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Volume 106, Issue 5, Pages 1053-1060


Fabiola Zambrano, M.Sc., Tessa Carrau, V.M., Ulrich Gärtner, Ph.D., Anika Seipp, M.T., Anja Taubert, Ph.D., Ricardo Felmer, Ph.D., Raúl Sanchez, Ph.D., Carlos Hermosilla, Ph.D.



To determine whether the human spermatozoon is a sufficient stimulus to trigger the release of neutrophil extracellular traps (NETs) in a time- and dose-dependent manner.


Experimental study.


University-based laboratory.


Semen samples from four men and blood samples from six healthy female donors.


Polymorphonuclear neutrophils (PMN) isolated from peripheral blood were incubated with fresh human spermatozoa for 60, 90, 120, and 180 minutes and at different PMN/sperm concentrations (1:1 [25 × 104], 1:3 [25 × 104:75 × 104], 1:6 [25 × 104:15 × 105], 1:18 [25 × 104:45 × 105]).

Main Outcome Measure(s)

During coincubation of PMN/sperm, the release of NETs was measured by PicoGreen. Immunofluorescence for histone H3, neutrophil elastase (NE), and myeloperoxidase (MPO) was performed. Different NETs inhibitors were tested: diphenylene iodonium, Suc-Ala- Ala-Pro-Val chloromethyl ketone (CMK), and 4-aminobenzoic acid hydrazide (ABAH) inhibitors of NADPH oxidase, NE, and MPO. Progressive mobility was assessed at increasing doses of neutrophils (1:18 [25 × 104:45 × 105], 6:18 [15 × 105:45 × 105], 9:18 [252 × 104:45 × 105]).


The quantity of NETs increased at the ratio of 1:6 after 2 hours and continued to increase subsequently. A ratio of 1:18 showed significant increases in NETs production at all times. Assessment of the inhibitors showed that CMK and ABAH inhibit NETs formation. Scanning and transmission electron microphotographs and immunofluorescence confirmed NETs formation induced by the spermatozoa. After 1 hour, progressive motility diminished in the two groups with the highest proportion of neutrophils and after 2 hours in all groups exposed to neutrophils.


We show that the stimulus of the human spermatozoon triggers the release of NETs; this response is dose dependent and increases with exposure time. The motility of affected spermatozoa diminishes, suggesting that this interaction on a larger scale would decrease the probability of successful fertilization.

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Fertility and Sterility

Editorial Office, American Society for Reproductive Medicine

Fertility and Sterility® is an international journal for obstetricians, gynecologists, reproductive endocrinologists, urologists, basic scientists and others who treat and investigate problems of infertility and human reproductive disorders.