27 INTRODUCTION The fast growing of nanotechnology led to an increased of the industrial production of nanoparticles (NPs). They have dimensions below 100 nm and they seem to have different physical and chemical properties from their bulk materials. Their use have been proposed for industrial and biomedical applications. NPs are attrac- tive for medical purposes for their important and unique features, such as their surface to mass ratio that is much larger than that of other particles, their quan- tum properties and their ability to adsorb and carry other compounds [1]. However, despite the wide application of nanomateri- als, there is a serious lack of information concerning the impact of NPs on human health and on the envi- ronment. Our interest was focussed on the reproductive field where studies on the effects of NPs on ejaculated sperm are rather limited. Wiwanitkit et al. [2] showed that sperm motility was affected when human spermatozoa were incubated with gold NPs, which were also able to penetrate in the sperm head and tail. On the other hand, Makhluf et al. [3] demonstrated that the motility and the ability to undergo the acro- some reaction of bovine spermatozoa were not affected by the presence of the magnetite NPs. The same group proved that magnetite NPs conjugated to anti-protein kinase C (PKC) antibody can be utilised for PKC lo- calization and inhibition of its function in sperm cells [4]. Although in vivo testing will continue to provide the most relevant information on human hazards, there is an economic and ethical impetus to minimize the bur- den of animal testing for rapid and validated alterna- tive models of in vitro cytotoxicity [5]. Correspondence to: Giulia Collodel Department of Biomedical Sciences, Section of Applied Biology, University of Siena Policlinico “Santa Maria alle Scotte”, Viale Bracci 2, 53100 Siena, Italy Phone: (+39) 0577 233504. e-mail: giulia.collodel@unisi.it IN VITRO EFFECT OF SILVER ENGINEERED NANOPARTICLES ON HUMAN SPERMATOZOA Terzuoli G. 1 , Iacoponi F. 1 , Moretti E. 1,2 , Renieri T. 1,2 , Baldi G. 3 , Collodel G. 1,2 1 Department of Biomedical Sciences, Applied Biology Section, University of Siena 2 Interdepartmental Centre for Research and Therapy of Male Infertility, University of Siena 3 Cericol Colorobbia Research Center, via Pietramarina, 123 Sovigliana Vinci, 50053 Firenze Abstract. Silver (Ag) NPs are among the most commercialized NPs due to their antimicrobial potential. They are highly at- tractive for possible applications in manufacture of medical device. However there is a serious lack of information concerning their impact on the human health and the environment. Moreover studies on the effects of NPs on ejaculated sperm are rather limited. For these reasons our study explored the in vitro effects of Ag NPs on human ejaculated spermatozoa. Ag NPs have been produced, characterized, and furnished by Colorobbia Industry, Sovigliana (Vinci, Florence, Italy). Aliquots of total semen were incubated at 37°C for 60 minutes (min) and 120 min at the concentration of 125 μM, 250 μM, and 500 μM of engineered Ag NPs. The control was represented by specimens of semen samples treated with the same procedure without NPs. After the incubations, sperm motility was evaluated following WHO guidelines and sperm viability was eval- uated by Eosin Y test. At the end of incubation with Ag NPs the samples were processed by a Field Emission Gun-based Scanning Transmition Electron Microscope/ Energy Dispertion Spectrometry (STEM/EDS). We observed that sperm motility percentage was significantly reduced in semen samples treated with 125 µM, 250 µM and 500 µM of Ag NPs after 60 min and 120 min of incubation respect to controls (P<0.001; P<0.01, 125 µM at 60 min). Sperm viability percentage significantly decreased in a progressive manner after 125 µM (P<0.05), 250 µM (P<0.05) and 500 µM (P<0.001) Ag NPs incubation at 60 min and 120 min. We did not find any significant difference between the values assessed after 60 min of NPs incubation and those estimated after 120 min of incubation. In the control samples, the sperm motility and the sperm viability percentages significantly decreased after 120 min of incubation (P<0.001) respect to the basal values. Ag NPs were undetectable in all treated samples by STEM/EDS. These in vitro results show a decline in sperm motility and viability in even at the lowest concentration used and the cytotoxic effect occurs in a dose dependent manner. It is noteworthy that in each experiment, for each concentration of NPs used, the percentage of sperm viability was always higher than the percentage of sperm motility; it means that spermatozoa were viable but immotile. Moreover Ag NPs was undetectable in all the treated samples by STEM/EDS analysis. We may hypothesize that Ag NPs, under aqueous conditions, release Ag+ that could damage sperm membrane and/or penetrate inside the cells and interfere with disulphide bonds of proteins of the periassonemal structures of the sperm tail. Keywords: silver nanoparticles, human sperm, sperm motility, sperm viability. ARTICLES