Article Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa

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1 RBMOnline - Vol 18. No Reproductive BioMedicine Online; on web 26 November 2008 Article Effect of ntioxidnt supplementtion of cryopreservtion medium on post-thw integrity of humn spermtozo Ms Ktrin Tylor gined her Bchelor of Science degree with first clss honours mjoring in Humn Biology from Edith Cown University Fculty of Computing, Helth nd Science, Joondlup Western Austrli in She currently holds the position of Senior Embryologist t Concept Fertility Centre, where she specilises in clinicl humn embryology nd spermtozo DNA frgmenttion. Her current reserch interest is in the role of geing, oxidtive stress nd DNA frgmenttion in mle infertility. Ms Ktrin Tylor Ktrin Tylor 1,2, Peter Roberts 2, Kthy Snders 3, Peter Burton 1,2,4 1 Concept Fertility Centre, King Edwrd Memoril Hospitl, 374 Bgot Rd, Subico, Western Austrli, 6009; 2 School of Exercise nd Helth Science, Edith Cown University, 100 Joondlup Drive, Joondlup, Western Austrli, 6027; 3 School of Antomy nd Humn Biology, The University of Western Austrli, 35 Stirling Highwy, Crwley, Western Austrli, Correspondence: Tel: ; e-mil: pete@conceptfert.com.u Abstrct This study exmined whether the ddition of n ntioxidnt to cryopreservtion medium could improve the post-thw integrity of cryopreserved humn spermtozo, prticulrly from men with bnorml semen prmeters. Semen smples were collected by msturbtion nd ssessed following WHO stndrds. Norml (n = 23) nd bnorml (n = 20) smples were divided into three liquots prior to cryopreservtion. The first liquot remined untreted nd ws mixed with cryopreservtion medium (in-house 1:1). The second nd third liquots were mixed with cryopreservtion medium contining either 100 mol or 200 mol vitmin E nlogue. Smples were frozen t 10 C per minute to 80 C, then plunged into liquid nitrogen. Thwed smples were ssessed for motility, vitlity nd DNA integrity. Split-plot repeted-mesures ANOVA ws used to ssess within-subject (dose) nd between-group (norml/bnorml) differences in post-thw motility index, vitlity stining nd DNA frgmenttion. Vitmin E dose ws significntly ssocited with post-thw motility (P = 0.041) nd the pttern of response cross doses ws similr for norml nd bnorml groups. Post-thw motility ws significntly improved by the ddition of 200 mol vitmin E (P = 0.006), but neither vitlity nor sperm DNA frgmenttion were ltered. These results suggest tht the ddition of vitmin E to cryopreservtion medium improves post-thw motility. Keywords: ntioxidnts, cryopreservtion, humn sperm, oxidtive stress, vitmin E Introduction 184 Over the lst 20 yers, considerble reserch hs been undertken to improve semen freezing protocols (reviewed in Royere et l., 1996; McLughlin, 2001, 2002). However, exposure to subzero tempertures nd re-wrming cn still result in the loss of structurl integrity nd functionl cpbilities of t lest 50% of the spermtozo (Critser et l., 1987; McLughlin et l., 1992; Go et l., 1993; McLughlin, 2001; Woods et l., 2004). Survivl rtes of cryopreserved humn spermtozo pper to be further compromised in semen smples from oligozoospermic men (Lin et l., 1998; Hughes et l., 1999; Counsel et l., 2004). Spermtozoon injury s result of cryopreservtion reduces post-thw motility nd vibility by disrupting the integrity of the crosoml nd plsm membrnes, inducing biochemicl ltertions nd by decresing DNA stbility (Royere et l., 1996; Pen et l., 2004). Cryopreservtion cn induce n incresed rte of lipid peroxidtion in the sperm plsm membrne cusing n overll increse in the concentrtion of oxygen rdicls in the smple (Bumber et l., 2003). Exposure to high rective oxygen species (ROS) concentrtions cn result in the disruption of mitochondril nd plsm membrnes, cuse chromosoml 2009 Published by Reproductive Helthcre Ltd, Duck End Frm, Dry Dryton, Cmbridge CB23 8DB, UK

2 Article - Antioxidnts nd cryopreservtion of humn spermtozo - K Tylor et l. nd DNA frgmenttion nd bring bout reduction in sperm motility (Prinud et l., 1997; Aitken nd Krusz, 2001; Gil- Guzmn et l., 2001; Anzr et l., 2002; Bumber et l., 2003), processes tht pper to be compounded by the poor semen prmeters of oligozoospermic smples (Aitken et l., 1998; Gil-Guzmn et l., 2001; Allmneni et l., 2005). Mny studies hve identified correltion between excessive oxidtive stress nd mle subfertility (reviewed Agrwl et l., 2004, 2006). It is possible tht dvncing ge is responsible for some of the increse in ROS concentrtions observed in subfertile popultions since it hs recently been shown in the rodent model tht spermtozo from older mles hs incresed ROS nd lipid peroxidtion, suggesting reduced cpcity to cope with oxidtive stress (Weir nd Robire, 2007). Advncing ge is lso ssocited with incresed sperm DNA frgmenttion (Schmid et l., 2007; Vgnini et l., 2007). The dverse effects of oxidtion cn be reduced by ntioxidnts tht re produced in the germinl epitheli, cn be found within spermtozo nd re present s component of seminl plsm (Agrwl et l., 2004). A blnce between ROS production nd seminl ntioxidnts normlly exists; however, the effects of endogenous ntioxidnts in poor semen smples re often diminished while the concentrtion of ROS is bnormlly high (Aitken et l., 1998). To overcome the dverse effects of sperm oxidtive stress, ntioxidnts hve been used either by supplementing the diet or the culture medium in vitro (Agrwl et l., 2004; Ebisch et l., 2007). Vitmin E, chin-breking ntioxidnt present in humn semen (Therond et l., 1996) nd the sperm plsm membrnes (Bolle et l., 2002), hs been shown to hve protective effect on sperm motility by suppressing lipid peroxidtion (Aitken nd Clrkson, 1988; Aitken et l., 1989). Therefore, vitmin E supplementtion of sperm cryopreservtion medium my protect spermtozo from cryopreservtion-induced increses in ROS nd subsequent loss of motility. A study using bor spermtozo demonstrted tht vitmin E supplementtion of the cryopreservtion medium prior to cryopreservtion significntly improved spermtozoon post-thw survivl rte nd motion prmeters (Pen et l., 2004). In the bor, the ejculte is produced in two portions. The first contins the mjority of spermtozo, whilst the second portion is of reduced number nd of poorer qulity. Vitmin E supplementtion in the pre-freeze medium cused n incresed sperm survivl rte in the second portion but not in the first (Pen et l., 2004), rising the possibility tht vitmin E supplementtion of cryopreservtion medium my improve post-thw prmeters of frozen humn spermtozo, prticulrly from bnorml smples. In the humn, vitmin E supplementtion of cryopreservtion medium produced smll improvements in sperm motility in five out of 12 cses with wide vrition between smples observed (Askri et l., 1994), but whether the vrition in effect ws due to differences in sperm qulity ws not reported. The im of this study therefore ws to determine if the supplementtion of cryopreservtion medium with vitmin E could improve postthw integrity of cryopreserved humn spermtozo prticulrly from men with bnorml semen prmeters. Mterils nd methods Written pprovl for this study ws grnted by the King Edwrd Memoril Hospitl Internl Ethics Committee nd by the Edith Cown University Fculty of Computing, Helth nd Science Ethics Sub-Committee for the Conduct of Humn Reserch. This study utilized semen remining fter routine semen nlysis t Concept Fertility Clinic, Subico, Western Austrli. In totl, 43 semen smples from 43 unselected ptients were collected by msturbtion into sterile continers. Ptient consent to use surplus semen ws obtined prior to use. Normozoospermi (n = 23) nd bnorml (n = 20) were clssified using World Helth Orgniztion (WHO) stndrds (World Helth Orgniztion, 1999). Following liquefction nd preliminry ssessment, 0.6 ml of ech semen smple ws liquoted into three 12-ml Flcon round bottom tubes (Flcon, Becton Dickinson Lbwre, USA). The first semen frction ws mixed with n equl volume (1:1) of semen cryopreservtion medium (glycerol 15%, NCl 5.8 g/l, KCl 0.4 g/l, C lctte 0.76 g/l, NHCO g/l, NH 2 PO g/l, MgSO g/l, HEPES 4.77 g/l, glucose 8.59 g/l, fructose 8.59 g/l, glycine 10 g/l, streptomycin 0.05 g/l, penicillin 0.05 g/l, humn serum lbumin 5%). The sme btch of cryopreservtion medium ws used for ll smples used in this study. The second semen frction ws mixed with n equl volume of cryopreservtion medium contining 100 mol of wter soluble vitmin E nlogue (6-hydroxy-2,5,7,8-tetrmethylchromon-2 crboxylic cid), Trolox (Sigm-Aldrich, Tyreso, Sweden), nd the third frction ws mixed with n equl volume of cryopreservtion medium contining 200 mol of vitmin E. The semen preprtions were drwn by syringe into lbelled 0.5 ml semen freezing strws (Genetics Austrli, Austrli) nd plugged (Poudre de Bouchge, IMV Technologies, Frnce). Two strws were prepred from ech frction. To stndrdize the pre freeze exposure to vitmin E, the smples were cooled immeditely fter ddition of the cryopreservtion medium in Plnr Kryo 10 control rte-freezer (Plnr, UK) t 10 C/min to temperture of 80 C prior to rpid plunging into liquid nitrogen ( 196 C). The smples were then stored in liquid nitrogen until post-thw nlysis. To ssess post-thw sperm prmeters (motility index, vitlity nd sperm DNA frgmenttion), strws were removed from liquid nitrogen nd llowed to thw t room temperture. The post-thw nlysis ws crried out by one embryologist nd the smples were coded so tht the embryologist ws blind to the experimentl groups. The motility index for ech frction ws then clculted using the formul: totl post-thw motility/ totl pre-freeze motility 100 (Hughes et l., 1999). Due to strw dmge during processing, the motility index of ech frction could not be ssessed for two subjects (one norml; one bnorml). Therefore, 22 norml subjects nd 19 subjects with bnorml semen prmeters were included in the sttisticl nlysis for motility index. To ssess post-thw vitlity, the eosin nigrosin dye exclusion stining ssy ws used s previously described (Counsel et l., 2004). Briefly, one drop of the thwed spermtozo ws mixed with two drops of 1% eosin stin (BDH Lbortory Supplies, UK). After 30 s, three drops of 10% nigrosin (BDH) 185

3 Article - Antioxidnts nd cryopreservtion of humn spermtozo - K Tylor et l. 186 were dded to ech solution. A drop of ech frction ws smered onto glss microscope slides nd llowed to ir-dry. The smers were ssessed by oil immersion light microscopy t 1000 mgnifiction. Live spermtozo pper white whilst ded spermtozo with disrupted membrnes hve tken up the eosin stin nd pper red. Vitlity ws quntified by counting minimum of 200 spermtozo on ech slide nd the proportion of live spermtozo expressed s percentge (norml n = 23; bnorml n = 20). To ssess DNA frgmenttion, 10 l of the sperm suspension from ech of the three frctions ws smered over silnized glss microscope slides (Superfrost Plus, Menzel-Glser, Brunschweig), nd llowed to ir-dry. The cells were fixed in 3:1 rtio methnol/cetic cid solution t room temperture for 5 min, then wshed in phosphte-buffered sline (PBS, Amber Scientific, Austrli) for 5 min. Spermtozo with DNA strnd breks were identified using the TdT (terminl deoxynucleotidyl trnsferse)-medited dudp nick-end lbelling (TUNEL) ssy (ApopTg Plus Peroxidse In situ Apoptosis Detection Kit, S7101; Chemicon Interntionl, Austrli). For positive control slides, 75 l of 0.2 g/ml DNse I solution ws used to induce sperm nucler DNA dmge through enzymtic digestion fter cell permebiliztion in PBS 0.1% Triton X-100. The DNse exposed slides were incubted t 37 C for 10 min in humidified chmber. The slides were then wshed in PBS 2 3 times for 5 min before resuming the sperm TUNEL protocol. Negtive control slides were not exposed to the terminl deoxynucleotidyl trnsferse substrte nd were treted with the Apoptg rection buffer only. DNA-dmged spermtozo were visulized by exposing spermtozo to DiAmino Bednizine for 4 min. Undmged spermtozo were counterstined with methyl green (0.5%) for 10 min. Spermtozo were viewed under 1000 microscopy using n oil immersion objective lens. Spermtozo tht hd entered the poptotic pthwy hd brown stined nuclei. Unffected cells were ple green. The percentge of DNA dmged spermtozo ws ssessed by counting 400 spermtozo on ech slide for ech of the three frctions (norml n = 6; bnorml n = 12). Sttisticl nlysis Independent smple t-tests were used to ssess differences between norml nd bnorml pre-freeze semen prmeters nd mle ge. To exmine the effects of vitmin E supplementtion on post-thw motility index, vitlity stining nd DNA frgmenttion in norml nd bnorml smples, mixed within-subjects (vitmin E dose) between-subjects (norml/ bnorml pre-freeze semen prmeters) nlysis of vrince, lso known s split-plot repeted-mesures ANOVA, ws used (Tbchnick nd Fidell, 2001). Given the ssocition between ge nd ROS concentrtions nd DNA dmge reported in the literture, ge ws included s covrite (continuous betweensubject vrible) in ech nlysis. Where significnt interction of within- nd between-subjects vribles occurred (ge nd vitmin E dose), follow-up univrite repeted-mesures ANOVA ws used to exmine the source of the interction (Tbchnick nd Fidell, 2001). For ll repeted-mesures nlyses, Greenhouse Geisser degrees of freedom corrections were used if the ssumption of homogeneity of covrince ws violted (Tbchnick nd Fidell, 2001). Results Semen prmeters of the smples used in this study re presented in Tble 1. Individul smples were clssified s norml (23) or bnorml (20). Individul smples from the bnorml group were clssified oligozoospermic (n = 6), tertozoospermic (n = 9), oligotertozoospermic (n = 3) nd oligosthenotertozoospermic (n = 2). Totl sperm concentrtion, motile sperm concentrtion nd morphologicl normlity were significntly higher (P < 0.001), nd ejculte volume ws significntly lower (P < 0.005) in the normozoospermic smples compred with bnorml smples. Post-thw motility index There ws no significnt difference in verge post-thw motility between norml nd bnorml groups (F [1,38] = 2.69). However, there ws significnt min effect of vitmin E dose (F [2,76] = 3.33, P = 0.041), with pirwise comprisons indicting tht post-thw motility ws significntly improved over control in the liquots supplemented with 200 mol vitmin E (P = 0.006, lest significnt difference [LSD] sttistic; Figure 1). The pttern of response to the vitmin E tretment ws similr for men with norml nd bnorml semen prmeters (group dose F [2,76] = 1.14). Age ws not independently ssocited with post-thw motility, but the effect of vitmin E dose vried by ge (ge dose F [2,76] = 3.95, P = 0.023). To investigte the source of the interction, seprte univrite repeted-mesures ANOVAs were crried out mongst men ged <40 yers (n = 27; norml = 14; bnorml = 13) nd men ged 40 yers (n = 14; norml n = 8; bnorml n = 6). The ANOVA for the men ged 40 yers ws significnt (F [2,26] = 5.27, P = 0.012) nd pirwise comprison showed tht the motility index ws significntly improved over control by the ddition of 200 of vitmin E (P = 0.003, LSD sttistic; Figure 2). In contrst, there ws no effect of vitmin E dose mongst men ged under 40 yers (F [2, 52] = 1.04). Post-thw vitlity No significnt differences in post-thw vitlity were observed cross the vitmin E tretment groups (norml smples: control 27.48%, 100 mol 28.06%, 200 mol 30.05%; bnorml smples: control 19.26%, 100 mol 20.31%, 200 mol 19.44%; F [2,80] = 0.25). However, post-thw vitlity ws significntly lower in the bnorml smples (F [1,40] = 11.53, P = 0.002) compred with the norml smples. There were no significnt effects of ge or significnt interctions between dose, group nd ge. Post-thw DNA frgmenttion There ws no significnt effect of vitmin E supplementtion on post thw DNA frgmenttion (norml smples: control 24.87%, 100 mol 22.83%, 200 mol 24.15%; bnorml smples: control 34.17%, 100 mol 38.21%, 200 mol 41.11%; F [1.45,21.8] = 1.16). DNA frgmenttion ws significntly higher in bnorml smples (F [1,15] = 6.426, P = 0.023) compred with norml smples. There were no significnt ge or interction effects on post-thw DNA frgmenttion.

4 Article - Antioxidnts nd cryopreservtion of humn spermtozo - K Tylor et l. Tble 1. Chrcteristics of semen smples included in the study. Smples were clssified s norml or bnorml ccording to World Helth Orgniztion stndrds (World Helth Orgniztion, 1999). Prmeter Norml (n = 23) Abnorml (n = 20) Age (yers) 36.7 ± 1.4 (25 48) 36.6 ± 1.3 (29 51) Ejculte volume (ml) 3.6 ± 0.3 ( ) 5.0 ± 0.3 ( ) Totl sperm concentrtion ( 10 6 /ml) 71.8 ± 5.6 ( ) 33.7 ± 5.1 b ( ) Motile sperm concentrtion ( 10 6 /m) 53.5 ± 5.1 ( ) 20.6 ± 3.5 b ( ) Motility (% forwrd progression) 91.4 ± 1.3 (77 100) 90.5 ± 1.7 (83 100) Morphology (% norml) 27.4 ± 2.0 (17 47) 12.9 ± 1.4 b (5 7) Vlues re the men ± SEM (rnge). P < compred with norml semen smples, independent smples t-test. b P < compred with norml smples, independent smples t-test. 40 Norml Abnorml <40 yers >40 yers Motility index (%) Motility index (%) Control 100 μm 200 μm Vitmin E concentrtion 0 Control 100 μm 200 μm Vitmin E concentrtion Figure 1. Reltionship between vitmin E supplementtion, pre-freeze semen prmeters nd post-thw motility index. Split plot repeted mesures, 200μM compred with control, P= Figure 2. The effect of vitmin E dose on post-thw motility index for men ged <40 yers nd men ged 40 yers. Univrite repeted-mesures nlysis of vrince, 200μM compred with control mongst men 40 yers, P= Discussion The im of the present study ws to determine if the ddition of n ntioxidnt to the semen cryopreservtion medium could improve the post-thw integrity of cryopreserved humn spermtozo prticulrly from men with bnorml semen prmeters. It ws found tht vitmin E supplementtion of semen cryopreservtion medium t concentrtion of 200 mol significntly improved post-thw motility, but not vitlity or the degree of DNA frgmenttion. The positive effect of vitmin E supplementtion ws ssocited with men s ge, with smples from men over 40 yers of ge benefiting the most. Vitmin E supplementtion did not pper to preferentilly improve postthw integrity of bnorml smples. Cryopreservtion cn result in incresed lipid peroxidtion in humn spermtozo (Alvrez nd Storey, 1992) nd hs been shown to reduce ntioxidnt defences in bovine spermtozo (Bilodeu et l., 2000). Thus, the protective effect of vitmin E ddition on post-thw motility observed in the present study might be due to vitmin E suppression of lipid peroxidtion vi the sperm plsm membrne (Aitken nd Clrkson, 1988; Aitken et l., 1989; Verm nd Knwr, 1999). Vitmin E supplementtion hs lso been shown to inhibit lipid peroxidtion in humn erythrocytes (Jin et l., 2000). The positive effect of vitmin E on sperm motility observed in the present study is supported by smller previous study in humns. Askri et l. (1994) reported tht vitmin E supplementtion of cryopreservtion medium produced smll improvements in sperm motility in five out of 12 cses; however, wide vrition between smples ws observed. Whether the vrition ws ssocited with ge ws not reported. In the bor, vitmin E supplementtion of pre-freeze medium hs been shown to increse sperm survivl rte in the second portion of the ejculte where the sperm qulity is reduced but not in the first portion contining the good-qulity spermtozo (Pen et l., 2004). No difference in motility ws evident between smples with norml nd bnorml semen prmeters in this study, suggesting tht vitmin E does not preferentilly protect 187

5 Article - Antioxidnts nd cryopreservtion of humn spermtozo - K Tylor et l. 188 poor-qulity humn semen smples s it does in the bor. The positive effect of vitmin E on motility observed in the present study ws greter in semen smples from men over 40 yers of ge. This could be due to protection from incresed ROS concentrtions since it hs recently been shown in the rodent model tht spermtozo from older mles hd incresed ROS nd lipid peroxidtion, suggesting reduced cpcity to cope with oxidtive stress (Weir nd Robire, 2007). Whether there is n ge-relted increse in oxidtive stress in humn spermtozo is not cler. Advncing ge cn lso increse sperm DNA frgmenttion (Schmid et l., 2007; Vgnini et l., 2007), lthough this effect ws not evident in the present study, possibly due to the lower number of smples nlysed. In the present study, the ddition of vitmin E to the cryopreservtion medium did not ffect post-thw vitlity or DNA frgmenttion. Confirming previous study (Counsel et l., 2004) post-thw vitlity s mesured by eosin nigrosin exclusion stining ws lower in the bnorml semen smple. Although not ffected by the ddition of vitmin E to the cryopreservtion medium, post-thw DNA frgmenttion ws significntly higher in the bnorml smples compred with the norml smples. This finding is consistent with other studies showing higher degrees of DNA frgmenttion in bnorml smples (Schmid et l., 2007; Vgnini et l., 2007). Furthermore, DNA strnd breks hve been found to occur to significntly higher degree following cryopreservtion in smples from men with oligozoospermi (Host et l., 1999). In conclusion, vitmin E supplementtion of semen cryopreservtion medium t concentrtion of 200 mol significntly improved post-thw motility, but not vitlity or the degree of DNA frgmenttion. The positive effect of vitmin E supplementtion ws ssocited with men s ge, with smples from men over 40 yers of ge benefiting the most. Further exmintion of the possible interctions between ntioxidnts nd semen prmeters re wrrnted. References Agrwl A, Sid TM, Bediwy MA et l Oxidtive stress in n ssisted reproductive techniques setting. Fertility nd Sterility 86, Agrwl A, Nllell KP, Allmneni SS, Sid TM 2004 Role of ntioxidnts in tretment of mle infertility: n overview of the tretment. Reproductive BioMedicine Online 8, Aitken RJ, Clrkson JS 1988 Significnce of rective oxygen species nd ntioxidnts in defining the efficcy of sperm preprtion techniques. Journl of Andrology 6, Aitken RJ, Krusz C 2001 Oxidtive stress, DNA dmge nd the Y chromosome. Reproduction 122, Aitken, RJ, Gordon, E, Hrkiss et l Reltive impct of oxidtive stress on the functionl competence nd genomic integrity of humn spermtozo. Biology of Reproduction 59, Aitken RJ, Clrkson JS, Fishel S 1989 Genertion of rective oxygen species, lipid peroxidtion, nd humn sperm function. Biology of Reproduction 1, Allmneni SS, Argrwl A, Nllell KP et l Chrcteristion of oxidtive stress sttus by evlution of rective oxygen species levels in whole semen nd isolted spermtozo. Fertility nd Sterility 83, Alvrez JG, Storey BT 1992 Evidence of incresed lipid peroxidtive dmge nd loss of superoxide dismutse ctivity s mode of sublethl cryodmge to humn sperm during cryopreservtion. Journl of Andrology 13, Anzr M, He L, Buhr M et l Sperm poptosis in fresh nd cryopreserved bull semen detected by flow cytometry nd its reltionship with fertility. Biology of Reproduction 66, Askri HA, Check, JH, Peymer N, Bollendorf A 1994 Effect of nturl ntioxidnts tocopherol nd scorbic cids in mintennce of sperm ctivity during freeze-thw process. Archives of Andrology 33, Bumber J, Bll BA, Linfor JJ, Meyers SA 2003 Rective oxygen species nd cryopreservtion promote DNA frgmenttion in equine spermtozo. 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6 Article - Antioxidnts nd cryopreservtion of humn spermtozo - K Tylor et l. Royere D, Brthelemy C, Hmmh S, Lnsc J 1996 Cryopreservtion of spermtozo: 1996 review. Humn Reproduction Updte 2, Schmid TE, Eskenzi B, Bumgrtner A et l The effects of mle ge on sperm DNA dmge in helthy non-smokers. Humn Reproduction 22, Tbchnick BG, Fidell LS 2001 Using Multivrite Sttistics 4th edn, Allyn nd Bcon, Needhm Heights, USA. Therond P, Auger J, Legrnd A, Jounnet P 1996 lph-tocopherol inhumn spermtozo nd seminl plsm: reltionship with motility, ntioxidnt enzymes nd leukocytes. Moleculr Humn Reproduction 2, Vgnini L, Bruffi RLR, Muri AL et l The effects of mle ge on sperm DNA dmge in n infertile popultion. Reproductive BioMedicine Online 15, Verm A, Knwr KC 1999 Effect of vitmin E on humn sperm motility nd lipid peroxidtion in vitro. Asin Journl of Andrology 1, Weir CP, Robire B 2007 Spermtozo hve decresed ntioxidnt enzymtic cpcity nd incresed rective oxygen species production during ging in the brown Norwy rt. Journl of Andrology 28, Woods E, Benson J, Agc Y, Critser J 2004 Fundmentl cryobiology of reproductive cells nd tissues. Cryobiology 48, World Helth Orgniztion 1999 WHO Lbortory Mnul for the Exmintion of Humn Semen nd Sperm-Cervicl Mucus Interction 4th edn. Cmbridge University Press, Cmbridge. Declrtion: The uthors report no finncil or commercil conflicts of interest. Received 2 April 2008; refereed 27 My 2008; ccepted 27 August