Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility Fabiola Zambrano, M.Sc., Tessa.

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Leukocytes coincubated with human sperm trigger classic neutrophil extracellular traps formation, reducing sperm motility 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. Fertility and Sterility Volume 106, Issue 5, Pages 1053-1060.e1 (October 2016) DOI: 10.1016/j.fertnstert.2016.06.005 Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Visualization of human spermatozoa-triggered NETs by SEM and TEM. NETs induced by vital human spermatozoa after 2 hours of exposure to human PMN. SEM analysis shows (A-D) the activation of PMN in the presence of human spermatozoa. Spermatozoa-triggered NETs formation and the capture of sperm by NETs structures. White arrows indicate NETs structures, red arrows indicate PMN, and yellow arrows indicate sperm being trapped by NETs. TEM (E and F) shows the activation of PMN by human sperm as the surface becomes rough and forms foldings and crinkles: (E) sperm phagocytosis and (F) completely covered sperm by NET-like extrusions. White arrows indicate NET-like extrusions, red arrows indicate intracellular sperm within PMN after phagocytosis, and yellow arrows indicate a sperm being entrapped by thicker NET-like extrusion (×98.500). Fertility and Sterility 2016 106, 1053-1060.e1DOI: (10.1016/j.fertnstert.2016.06.005) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Representative immunofluorescence images of PMN exposed to sperm. Coincubation PMN/sperm ratio 1:6 for 2 hours. Phase contrast (A, E, and I), blue fluorescence stains the nuclei of cells (DAPI, B, F, and J), green fluorescence (Alexafluor 488) shows different components of NETs, (C) HIS (histone H3), (G) MPO, and (K) NE. Colocalization areas are shown in panels D, H, and L by mixing both channels (merge). Fertility and Sterility 2016 106, 1053-1060.e1DOI: (10.1016/j.fertnstert.2016.06.005) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect in time on the progressive motility of human sperm of incubation of PMN/sperm with different ratios (1:18, 6:18, 9:18). Data are shown as percentages of progressively motile sperm (progressive motility [%]). Graph with three biological replicas. The results correspond to the mean ± SD. Differences were regarded as significant (*, ***) at a level of P<.001. Fertility and Sterility 2016 106, 1053-1060.e1DOI: (10.1016/j.fertnstert.2016.06.005) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Coincubation PMN/sperm ratio 1:6, for 2 hours. (A) Blue fluorescence stains the DNA of nuclei cells and NETs (white arrows) of DAPI (NETs trapping sperm), (B and C), green fluorescence (Alexa fluor 488) shows different components of NETs: (B) green arrows, MPO; (C) red arrow, colocalization of DNA and NE. Fertility and Sterility 2016 106, 1053-1060.e1DOI: (10.1016/j.fertnstert.2016.06.005) Copyright © 2016 American Society for Reproductive Medicine Terms and Conditions