Advertisement

Perfluoro-octanoic acid impairs sperm motility through the alteration of plasma membrane

  • I. Šabović
  • I. Cosci
  • L. De Toni
  • A. Ferramosca
  • M. Stornaiuolo
  • A. Di Nisio
  • S. Dall’Acqua
  • A. Garolla
  • C. ForestaEmail author
Original Article
  • 45 Downloads

Abstract

Context

Perfluoroalkyl-substances (PFAS) are chemical additives considered harmful for humans. We recently showed that accumulation of perfluoro-octanoic acid (PFOA) in human semen of exposed subjects was associated with altered motility parameters of sperm cells, suggesting direct toxicity.

Objectives

To determine whether direct exposure of human spermatozoa to PFOA was associated to impairment of cell function.

Patients and methods

Spermatozoa isolated from semen samples of ten normozoospermic healthy donors were exposed up to 2 h to PFOA, at concentrations from 0.1 to 10 ng/mL. Viability and motility parameters were evaluated by Sperm Class Analyser. Cell respiratory function was assessed by both mitochondrial probe JC-1 and respiratory control ratio (RCR) determination. Sperm accumulation of PFOA was quantified by liquid chromatography–mass spectrometry. Expression of organic ion-transporters OATP1 and SLCO1B2 was assessed by immunofluorescence and respective role in PFOA accumulation was evaluated by either blockade with probenecid or membrane scavenging through β-cyclodextrin (β-CD). Plasma membrane fluidity and electrochemical potential (ΔΨp) were evaluated, respectively, with Merocyanine-540 and Di-3-ANEPPDHQ fluorescent probes.

Results

Compared to untreated controls, a threefold increase of the percentage of non-motile sperms was observed after 2 h of exposure to PFOA regardless of the concentration of PFOA, whilst RCR was significantly reduced. Only scavenging with β-CD was effective in reducing PFOA accumulation, suggesting membrane involvement. Altered membrane fluidity, reduced ΔΨp and sperm motility loss associated with exposure to PFOA were reverted by β-CD treatment.

Conclusion

PFOA alters human sperm motility through plasma-membrane disruption, an effect recovered by incubation with β-CD.

Keywords

Sperm motility Liquid chromatography–mass spectrometry Probenecid Membrane fluidity 

Notes

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Institutional Ethics Committee of the Hospital of Padova, Italy, (protocol number 2208P and successive amendments). The investigation was performed according to the principles of the Declaration of Helsinki.

Informed consent

All patients provided signed informed consent at enrollment.

Supplementary material

40618_2019_1152_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 14 kb)

References

  1. 1.
    ATSDR (2008) Agency for toxic substances and disease registry. In: Draft Toxicological Profile for Perfluoroalkyls, AtlantaGoogle Scholar
  2. 2.
    Buck RC, Franklin J, Berger U, Conder JM, Cousins IT, de Voogt P, Jensen AA, Kannan K, Mabury SA, van Leeuwen SP (2011) Perfluoroalkyl and polyfluoroalkyl substances in the environment: terminology, classification, and origins. Integr Env Assess Manag 7:513–541.  https://doi.org/10.1002/ieam.258 CrossRefGoogle Scholar
  3. 3.
    Mamsen LS, Björvang RD, Mucs D, Vinnars MT, Papadogiannakis N, Lindh CH, Andersen CY, Damdimopoulou P (2019) Concentrations of perfluoroalkyl substances (PFASs) in human embryonic and fetal organs from first, second, and third trimester pregnancies. Env Int 124:482–492.  https://doi.org/10.1016/j.envint.2019.01.010 CrossRefGoogle Scholar
  4. 4.
    Austin ME, Kasturi BS, Barber M, Kannan K, Mohan Kumar PS, Mohan Kumar SM (2003) Neuroendocrine effects of perfluorooctane sulfonate in rats. Env Health Perspect 111:1485–1489.  https://doi.org/10.1289/ehp.6128 CrossRefGoogle Scholar
  5. 5.
    Inoue K, Okada F, Ito R, Kato S, Sasaki S, Nakajima S, Uno A, Saijo Y, Sata F, Yoshimura Y,. Kishi R, Nakazawa H. (2004) Perfluorooctane sulfonate (PFOS) and related perfluorinated compounds in human maternal and cord blood samples: assessment of PFOS exposure in a susceptible population during pregnancy. Environ Health Perspect112(11):1204–1207.  https://doi.org/10.1289/ehp.6864 (PMID: 15289168)CrossRefGoogle Scholar
  6. 6.
    Fei C, McLaughlin JK, Tarone RE, Olsen J (2007) Perfluorinated chemicals and fetal growth: a study within the Danish National Birth Cohort. Env Health Perspect 115:1677–1682.  https://doi.org/10.1289/ehp.10506 CrossRefGoogle Scholar
  7. 7.
    Kim S, Choi K, Ji K, Seo J, Kho Y, Park J, Kim S, Park S, Hwang I, Jeon J, Yang H, Giesy JP (2011) Trans-placental transfer of thirteen perfluorinated compounds and relations with fetal thyroid hormones. Env Sci Technol 45:7465–7472.  https://doi.org/10.1021/es202408a CrossRefGoogle Scholar
  8. 8.
    Li N, Mruk DD, Chen H, Wong CKC, Lee WM, Cheng CY (2016) Rescue of perfluorooctanesulfonate (PFOS)–mediated Sertoli cell injury by overexpression of gap junction protein connexin 43. Sci Rep 6:29667.  https://doi.org/10.1038/srep29667 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Calafat AM, Wong LY, Kuklenyik Z, Reidy JA, Needham LL (2007) Polyfluoroalkyl chemicals in the US population: data from the National Health and Nutrition Examination Survey (NHANES) 2003–2004 and comparisons with NHANES 1999–2000. Env Health Perspect 115:1596–1602.  https://doi.org/10.1289/ehp.10598 CrossRefGoogle Scholar
  10. 10.
    EFSA (European Food Safety Authority) (2008) Perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts: scientific opinion of the panel on contaminants in the food chain. EFSA J 653:1–131.  https://doi.org/10.2903/j.efsa.2008.653 CrossRefGoogle Scholar
  11. 11.
    Fromme H, Tittlemier SA, Volkel W, Wilhelm M, Twardella D (2009) Perfluorinated compounds—exposure assessment for the general population in Western countries. Int J Hyg Env Health 212:239–270.  https://doi.org/10.1016/j.ijheh.2008.04.007 CrossRefGoogle Scholar
  12. 12.
    Roosens L, D’Hollander W, Bervoets L, Reynders H, Van Campenhout K, Cornelis C, Van Den Heuvel R, Koppen G, Covaci A (2010) Brominated flame retardants and perfluorinated chemicals, two groups of persistent contaminants in Belgian human blood and milk. Env Pollut 158:2546–2552.  https://doi.org/10.1016/j.envpol.2010.05.022 CrossRefGoogle Scholar
  13. 13.
    Stahl T, Mattern D, Brunn H (2011) Toxicology of perfluorinated compounds. Env Sci Eur 23:38.  https://doi.org/10.1186/2190-4715-23-38 CrossRefGoogle Scholar
  14. 14.
    Domingo JL (2012) Health risks of dietary exposure to perfluorinated compounds. Env Int 40:187–195.  https://doi.org/10.1016/j.envint.2011.08.001 CrossRefGoogle Scholar
  15. 15.
    Cornelis C, D’Hollander W, Roosens L, Covaci A, Smolders R, Van Den Heuvel R, Govarts E, Van Campenhout K, Reynders H, Bervoets L (2012) First assessment of population exposure to perfluorinated compounds in Flanders, Belgium. Chemosphere 86:308–314.  https://doi.org/10.1016/j.chemosphere.2011.10.034 CrossRefPubMedGoogle Scholar
  16. 16.
    Lindh CH, Rylander L, Toft G, Axmon A, Rignell-Hydbom A, Giwercman A, Pedersen HS, Góalczyk K, Ludwicki JK, Zvyezday V, Vermeulen R, Lenters V, Heederik D, Bonde JP, Jönsson BA (2012) Blood serum concentrations of perfluorinated compounds in men from Greenlandic Inuit and European populations. Chemosphere 88:1269–1275.  https://doi.org/10.1016/j.chemosphere.2012.03.049 CrossRefPubMedGoogle Scholar
  17. 17.
    Ding G, Xue H, Yao Z, Wang Y, Ge L, Zhang J, Cui F (2018) Occurrence and distribution of perfluoroalkyl substances (PFASs) in the water dissolved phase and suspended particulate matter of the Dalian Bay, China. Chemosphere 200:116–123.  https://doi.org/10.1016/j.chemosphere.2018.02.093 CrossRefPubMedGoogle Scholar
  18. 18.
    Di Nisio A, Sabovic I, Valente U, Tescari S, Rocca MS, Guidolin D, Dall’Acqua S, Acquasaliente L, Pozzi N, Plebani M, Garolla A, Foresta C (2019) Endocrine disruption of androgenic activity by perfluoroalkyl substances: clinical and experimental evidence. J Clin Endocrinol Metab 104:1259–1271.  https://doi.org/10.1210/jc.2018-01855 CrossRefPubMedGoogle Scholar
  19. 19.
    Qiao W, Zhang Y, Xie Z, Luo Y, Zhang X, Sang C, Xie S, Huang J (2019) Toxicity of perfluorooctane sulfonate on Phanerochaete chrysosporium: growth, pollutant degradation and transcriptomics. Ecotoxicol Env Saf 174:66–74.  https://doi.org/10.1016/j.ecoenv.2019.02.066 CrossRefGoogle Scholar
  20. 20.
    World Health Organization (2010) Department of Reproductive Health and Research. In: WHO laboratory manual for the examination and processing of human semen; Fifth edition. SwitzerlandGoogle Scholar
  21. 21.
    Zuccarello D, Ferlin A, Garolla A, Menegazzo M, Perilli L, Ambrosini G, Foresta C (2011) How the human spermatozoa sense the oocyte: a new role of SDF1-CXCR4 signalling. Int J Androl 34(6 Pt 2):e554–e565.  https://doi.org/10.1111/j.1365-2605.2011.01158.x CrossRefPubMedGoogle Scholar
  22. 22.
    Grami D, Rtibi K, Selmi S, Jridi M, Sebai H, Marzouki L, Šabovic I, Foresta C, De Toni L (2018) Aqueous extract of Eruca Sativa protects human spermatozoa from mitochondrial failure due to bisphenol A exposure. Reprod Toxicol 82:103–110.  https://doi.org/10.1016/j.reprotox.2018.10.008 CrossRefPubMedGoogle Scholar
  23. 23.
    Muratori M, Porazzi I, Luconi M, Marchiani S, Forti G, Baldi E (2004) AnnexinV binding and merocyanine staining fail to detect human sperm capacitation. J Androl 25:797–810.  https://doi.org/10.1002/j.1939-4640.2004.tb02858.x CrossRefPubMedGoogle Scholar
  24. 24.
    Ferramosca A, Focarelli R, Piomboni P, Coppola L, Zara V (2008) Oxygen uptake by mitochondria in demembranated human spermatozoa: a reliable tool for the evaluation of sperm respiratory efficiency. Int J Androl 31:337–345.  https://doi.org/10.1111/j.1365-2605.2007.00775.x CrossRefPubMedGoogle Scholar
  25. 25.
    Bedu-Addo K, Lefièvre L, Moseley FL, Barratt CL, Publicover SJ (2005) Bicarbonate and bovine serum albumin reversibly “switch” capacitation-induced events in human spermatozoa. Mol Hum Reprod 11:683–691.  https://doi.org/10.1093/molehr/gah226 CrossRefPubMedGoogle Scholar
  26. 26.
    Alonso CAI, Osycka-Salut CE, Castellano L, Cesari A, Di Siervi N, Mutto A, Johannisson A, Morrell JM, Davio C, Perez-Martinez S (2017) Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines. Mol Hum Reprod 23:521–534.  https://doi.org/10.1093/molehr/gax030 CrossRefPubMedGoogle Scholar
  27. 27.
    Mor AL, Kaminski TW, Karbowska M, Pawlak D (2018) New insight into organic anion transporters from the perspective of potentially important interactions and drugs toxicity. J Physiol Pharmacol.  https://doi.org/10.26402/jpp.2018.3.01 CrossRefPubMedGoogle Scholar
  28. 28.
    Han X, Nabb DL, Russell MH, Kennedy GL, Rickard RW (2012) Renal elimination of perfluorocarboxylates (PFCAs). Chem Res Toxicol 25(1):35–46.  https://doi.org/10.1021/tx200363w CrossRefPubMedGoogle Scholar
  29. 29.
    Vanden Heuvel JP, Davis JW 2nd, Sommers R, Peterson RE (1992) Renal excretion of perfluorooctanoic acid in male rats: inhibitory effect of testosterone. J Biochem Toxicol Spring 7:31–36CrossRefGoogle Scholar
  30. 30.
    Ylinen M, Hanhijärvi H, Jaakonaho J, Peura P (1989) Stimulation by oestradiol of the urinary excretion of perfluorooctanoic acid in the male rat. Pharmacol Toxicol 65:274–277.  https://doi.org/10.1111/j.1600-0773.1989.tb01172.x CrossRefPubMedGoogle Scholar
  31. 31.
    Takeda M, Khamdang S, Narikawa S, Kimura H, Kobayashi Y, Yamamoto T, Cha SH, Sekine T, Endou H (2002) Human organic anion transporters and human organic cation transporters mediate renal antiviral transport. J Pharmacol Exp Ther 300:918–924.  https://doi.org/10.1124/jpet.300.3.918 CrossRefPubMedGoogle Scholar
  32. 32.
    Kleszczyński K, Składanowski AC (2009) Mechanism of cytotoxic action of perfluorinated acids. I. alteration in plasma membrane potential and intracellular pH level. Toxicol Appl Pharmacol 234:300–305.  https://doi.org/10.1016/j.taap.2008.10.008 CrossRefPubMedGoogle Scholar
  33. 33.
    Nouhi S, Ahrens L, Campos Pereira H, Hughes AV, Campana M, Gutfreund P (2018) Interactions of perfluoroalkyl substances with a phospholipid bilayer studied by neutron reflectometry. J Colloid Interface Sci 511:474–481.  https://doi.org/10.1016/j.jcis.2017.09.102 CrossRefPubMedGoogle Scholar
  34. 34.
    Fitzgerald NJM, Wargenau A, Sorenson C, Pedersen J, Tufenkji N, Novak PJ, Simcik MF (2018) Partitioning and accumulation of perfluoroalkyl substances in model lipid bilayers and bacteria. Environ Sci Technol 52:10433–10440.  https://doi.org/10.1021/acs.est.8b02912 CrossRefPubMedGoogle Scholar
  35. 35.
    Coisne C, Tilloy S, Monflier E, Wils D, Fenart L, Gosselet F (2016) Cyclodextrins as emerging therapeutic tools in the treatment of cholesterol-associated vascular and neurodegenerative diseases. Molecules 21:E1748.  https://doi.org/10.3390/molecules21121748 CrossRefGoogle Scholar
  36. 36.
    Weiss-Errico MJ, Miksovska J, O’Shea KE (2018) β-Cyclodextrin reverses binding of perfluorooctanoic acid to human serum albumin. Chem Res Toxicol 31:277–284.  https://doi.org/10.1021/acs.chemrestox.8b00002 CrossRefPubMedGoogle Scholar
  37. 37.
    Buffone MG, Brugo-Olmedo S, Calamera JC, Verstraeten SV, Urrutia F, Grippo L, Corbetta JP, Doncel GF (2006) Decreased protein tyrosine phosphorylation and membrane fluidity in spermatozoa from infertile men with varicocele. Mol Reprod Dev 73:1591–1599.  https://doi.org/10.1002/mrd.20611 CrossRefPubMedGoogle Scholar
  38. 38.
    Williamson P, Mattocks K, Schlegal RA (1983) Merocyanine 540, a fluorescent probe sensitive to lipid packaging. Biochim Biophys Acta 732:387–393.  https://doi.org/10.1016/0005-2736(83)90055-X CrossRefGoogle Scholar
  39. 39.
    Langner M, Hui SW (1993) Merocyanine interaction with phosphatidylcholine bilayers. Biochim Biophys Acta 1149:175–179.  https://doi.org/10.1016/0005-2736(93)90038-2 CrossRefPubMedGoogle Scholar
  40. 40.
    Rathi R, Colenbrander B, Bevers MM, Gadella BM (2001) Evaluation of in vitro capacitation of stallion spermatozoa. Biol Reprod 65:462–470.  https://doi.org/10.1095/biolreprod65.2.462 CrossRefPubMedGoogle Scholar
  41. 41.
    van Gestel RA, Helms JB, Brouwers JF, Gadella BM (2005) Effects of methyl-beta-cyclodextrin-mediated cholesterol depletion in porcine sperm compared to somatic cells. Mol Reprod Dev 72:386–395.  https://doi.org/10.1002/mrd.20351 CrossRefPubMedGoogle Scholar
  42. 42.
    Zavodnik IB, Lapshina EA, Palecz D, Bryszewska M (1996) The effects of palmitate on human erythrocyte membrane potential and osmotic stability. Scand J Clin Lab Invest 56:401–407.  https://doi.org/10.3109/00365519609088794 CrossRefPubMedGoogle Scholar
  43. 43.
    Tillman TS, Cascio M (2003) Effects of membrane lipids on ion channel structure and function. Cell Biochem Biophys 38:161–190.  https://doi.org/10.1385/CBB:38:2:161 CrossRefPubMedGoogle Scholar
  44. 44.
    Ritagliati C, Baro Graf C, Stival C, Krapf D (2018) Regulation mechanisms and implications of sperm membrane hyperpolarization. Mech Dev 154:33–43.  https://doi.org/10.1016/j.mod.2018.04.004 CrossRefPubMedGoogle Scholar
  45. 45.
    Jensen AA, Leffers H (2008) Emerging endocrine disrupters: perfluoroalkylated substances. Int J Androl 31:161–169.  https://doi.org/10.1111/j.1365-2605.2008.00870.x CrossRefPubMedGoogle Scholar
  46. 46.
    Chen MH, Ha EH, Wen TW, Su YN, Lien GW, Chen CY, Chen PC, Hsieh WS (2012) Perfluorinated compounds in umbilical cord blood and adverse birth outcomes. PLoS One 7:e42474.  https://doi.org/10.1371/journal.pone.0042474 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Giesy JP, Kannan K (2001) Global distribution of perfluorooctane sulfonate in wildlife. Environ Sci Technol 35:1339–1342CrossRefGoogle Scholar
  48. 48.
    Di Nisio A, Foresta C (2019) Water and soil pollution as determinant of water and food quality/contamination and its impact on male fertility. Reprod Biol Endocrinol. 17:4.  https://doi.org/10.1186/s12958-018-0449-4 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Kim SJ, Heo SH, Lee DS, Hwang IG, Lee YB, Cho HY (2016) Gender differences in pharmacokinetics and tissue distribution of 3 perfluoroalkyl and polyfluoroalkyl substances in rats. Food Chem Toxicol 97:243–255.  https://doi.org/10.1016/j.fct.2016.09.017 CrossRefPubMedGoogle Scholar
  50. 50.
    Lau C, Anitole K, Hodes C, Lai D, Pfahles-Hutchens A, Seed J (2007) Perfluoroalkyl acids: a review of monitoring and toxicological findings. Toxicol Sci 99:366–394.  https://doi.org/10.1093/toxsci/kfm128 CrossRefPubMedGoogle Scholar
  51. 51.
    Kudo N, Kawashima Y (2003) Toxicity and toxicokinetics of perfluorooctanoic acid in humans and animals. J Toxicol Sci 28:49–57.  https://doi.org/10.2131/jts.28.49 CrossRefPubMedGoogle Scholar
  52. 52.
    Kennedy GL Jr, Butenhoff JL, Olsen GW, O’Connor JC, Seacat AM, Perkins RG, Biegel LB, Murphy SR, Farrar DG (2004) The toxicology of perfluorooctanoate. Crit Rev Toxicol 34:351–384CrossRefGoogle Scholar
  53. 53.
    Joensen UN, Bossi R, Leffers H, Jensen AA, Skakkebaek NE, Jørgensen N (2009) Do perfluoroalkyl compounds impair human semen quality? Environ Health Perspect 117:923–927.  https://doi.org/10.1289/ehp.0800517 CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Governini L, Guerranti C, De Leo V, Boschi L, Luddi A, Gori M, Orvieto R, Piomboni P (2015) Chromosomal aneuploidies and DNA fragmentation of human spermatozoa from patients exposed to perfluorinated compounds. Andrologia. 47:1012–1019.  https://doi.org/10.1111/and.12371 CrossRefPubMedGoogle Scholar
  55. 55.
    Flesch FM, Gadella BM (2000) Dynamics of the mammalian sperm plasma membrane in the process of fertilization. Biochim Biophys Acta 1469:197–235.  https://doi.org/10.1016/S0304-4157(00)00018-6 CrossRefPubMedGoogle Scholar
  56. 56.
    Nikolopoulou M, Soucek DA, Vary JC (1985) Changes in the lipid content of boar sperm plasma membranes during epididymal maturation. Biochim Biophys Acta 815:486–498.  https://doi.org/10.1016/0005-2736(85)90377-3 CrossRefPubMedGoogle Scholar
  57. 57.
    Toshimori K (1998) Maturation of mammalian spermatozoa: modifications of the acrosome and plasma membrane leading to fertilization. Cell Tissue Res 293:177–187.  https://doi.org/10.1007/s004410051110 CrossRefPubMedGoogle Scholar
  58. 58.
    Haidl G, Opper C (1997) Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation. Hum Reprod 12:2720–2723.  https://doi.org/10.1093/humrep/12.12.2720 CrossRefPubMedGoogle Scholar
  59. 59.
    Vos JP, Lopes-Cardozo M, Gadella BM (1994) Metabolic and functional aspects of sulfogalactolipids. Biochim Biophys Acta 1211:125–149.  https://doi.org/10.1016/0005-2760(94)90262-3 CrossRefPubMedGoogle Scholar
  60. 60.
    Arbo MD, Altknecht LF, Cattani S, Braga WV, Peruzzi CP, Cestonaro LV, Göethel G, Durán N, Garcia SC (2019) In vitro cardiotoxicity evaluation of graphene oxide. Mutat Res 841:8–13.  https://doi.org/10.1016/j.mrgentox.2019.03.004 CrossRefPubMedGoogle Scholar
  61. 61.
    Stival C, Puga Molina Ldel C, Paudel B, Buffone MG, Visconti PE, Krapf D (2016) Sperm capacitation and acrosome reaction in mammalian sperm. Adv Anat Embryol Cell Biol 220:93–106.  https://doi.org/10.1007/978-3-319-30567-7_5 CrossRefPubMedGoogle Scholar
  62. 62.
    Lishko PV, Botchkina IL, Fedorenko A, Kirichok Y (2010) Acid extrusion from human spermatozoa is mediated by flagellar voltage-gated proton channel. Cell 140:327–337.  https://doi.org/10.1016/j.cell.2009.12.053 CrossRefPubMedGoogle Scholar
  63. 63.
    Lishko PV, Kirichok Y (2010) The role of Hv1 and CatSper channels in sperm activation. J Physiol 588(Pt 23):4667–4672.  https://doi.org/10.1113/jphysiol.2010.194142 CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    Foresta C, De Carlo E, Zorzi M, Rossato M, Finelli L (1990) Possible significance of seminal zinc on human spermatozoa functions. Acta Eur Fertil. 21:305–308PubMedGoogle Scholar
  65. 65.
    Costello LC, Liu Y, Franklin RB, Kennedy MC (1997) Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. J Biol Chem 272:28875–28881.  https://doi.org/10.1074/jbc.272.46.28875 CrossRefPubMedGoogle Scholar

Copyright information

© Italian Society of Endocrinology (SIE) 2019

Authors and Affiliations

  1. 1.Department of Medicine and Unit of Andrology and Reproductive MedicineUniversity of PadovaPaduaItaly
  2. 2.Familial Cancer ClinicVeneto Institute of Oncology (IOV-IRCCS)PaduaItaly
  3. 3.Department of Biological and Environmental Sciences and TechnologiesUniversity of SalentoLecceItaly
  4. 4.Department of Pharmaceutical and Pharmacological SciencesUniversity of PadovaPaduaItaly

Personalised recommendations