1465Vol. 11 | No. 02 | March- April 2014 |U R O LO G Y J O U R N A L Vitrification of Neat Semen Alters Sperm Pa- rameters and DNA Integrity Mohammad Ali Khalili, Maryam Adib, Iman Halvaei, Ali Nabi Corresponding Author: Maryam Adib, PhD Research and Clinical Center for In- fertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. Tell: +98 351 8247085 Fax: +98 351 8247087 E-mail: HYPERLINK "mailto:blueocean10221@yahoo. com" blueocean10221@yahoo.com Received March 2013 Accepted July 2013 Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. SEXUAL DYSFUNCTION AND INFERTILITY Purpose:‎Our‎aim‎was‎to‎evaluate‎the‎effect‎of‎neat‎semen‎vitrification‎on‎human‎sperm‎vital‎ parameters‎and‎DNA‎integrity‎in‎men‎with‎normal‎and‎abnormal‎sperm‎parameters.‎ Materials and Methods:‎Semen‎samples‎were‎17‎normozoospermic‎samples‎and‎17‎speci- mens‎with‎abnormal‎sperm‎parameters.‎Semen‎analysis‎was‎performed‎according‎to‎World‎ Health‎Organization‎(WHO)‎criteria.‎Then,‎the‎smear‎was‎provided‎from‎each‎sample‎and‎fixed‎ for‎terminal‎deoxynucleotidyl‎transferase‎dUTP‎nick‎end‎labeling‎(TUNEL)‎staining.‎Vitrifica- tion‎of‎neat‎semen‎was‎done‎by‎plunging‎cryoloops‎directly‎into‎liquid‎nitrogen‎and‎preserved‎ for‎7‎days.‎The‎samples‎were‎warmed‎and‎re-evaluated‎for‎sperm‎parameters‎as‎well‎as‎DNA‎ integrity.‎Besides,‎the‎correlation‎between‎sperm‎parameters‎and‎DNA‎fragmentation‎was‎as- sessed‎pre-‎and‎post‎vitrification. Results:‎Cryopreserved‎spermatozoa‎showed‎significant‎decrease‎in‎sperm‎motility,‎viability‎ and‎normal‎morphology‎after‎thawing‎in‎both‎normal‎and‎abnormal‎semen.‎Also,‎the‎rate‎of‎ sperm‎DNA‎fragmentation‎was‎significantly‎higher‎after‎vitrification‎compared‎to‎fresh‎sam- ples‎in‎normal‎(24.76‎±‎5.03‎and‎16.41‎±‎4.53,‎P‎=‎.002)‎and‎abnormal‎(34.29‎±‎10.02‎and‎ 23.5‎±‎8.31, P‎<‎.0001),‎respectively.‎There‎was‎negative‎correlation‎between‎sperm‎motility‎ and‎sperm‎DNA‎integrity‎in‎both‎groups‎after‎vitrification.‎ Conclusion:‎Vitrification‎of‎neat‎ejaculates‎has‎negative‎impact‎on‎sperm‎parameters‎as‎well‎ as‎DNA‎integrity,‎particularly‎among‎abnormal‎semen‎subjects.‎It‎is,‎therefore,‎recommend‎ to‎process‎semen‎samples‎and‎vitrify‎the‎sperm‎pellets.‎ Keywords:‎vitrification;‎humans;‎DNA‎damage;‎cryopreservation;‎methods;‎infertility;‎sper- matozoa;‎semen‎preservation..‎ 1466 | Sexual Dysfunction And Infertility INTRODUCTION Cryopreservation‎ of‎ human‎ spermatozoa‎ is‎ per-formed‎ routinely‎ in‎ assisted‎ reproductive‎ tech-nology‎ (ART)‎ program.‎ Sperm‎ bank‎ is‎ mainly‎ developed‎for‎men‎that‎are‎undergoing‎chemotherapy/radi- otherapy,‎ART‎treatment‎cycles,‎or‎have‎ejaculation‎abnor- malities‎and‎azoospermia.‎It‎has‎been‎reported‎that‎sperm‎ cryopreservation‎might‎have‎several‎impacts‎on‎sperm‎cell,‎ such‎ as‎ excessive‎ dehydration,‎ damage‎ to‎ plasma‎ mem- brane‎ and‎ acrosome‎ cap,‎ mitochondria‎ injury,‎ apoptosis‎ and‎ sperm‎ DNA‎ fragmentation.(1-3)‎ There‎ are‎ currently‎ three‎methods‎of‎cryopreservation‎namely:‎slow‎freezing,‎ rapid‎ freezing‎ and‎ vitrification.‎ The‎ first‎ two‎ techniques‎ have‎ been‎ in‎ practice‎ for‎ decades.‎ However,‎ they‎ have‎ some‎drawbacks,‎such‎as‎requiring‎expensive‎equipment,‎ are‎time‎and‎labor‎consuming‎and‎have‎limited‎efficacy.(4) Vitrification‎is‎the‎freezing‎method‎based‎on‎ultra-rapid‎cool- ing‎of‎water‎to‎glassy‎state‎at‎the‎high‎viscosity‎with‎no‎in- tracellular‎ ice‎ formation.(5)‎ Vitrification‎ of‎ sperm‎ freezing‎ was‎first‎introduced‎by‎the‎Isachenko’s‎group,‎in‎which‎the‎ samples‎were‎directly‎and‎quickly‎plunged‎into‎the‎liquid‎ni- trogen‎(LN).(6,7)‎Sperm‎vitrification‎is‎fast,‎simple‎and‎more‎ cost‎effective‎compared‎to‎slow‎freezing.‎Also,‎vitrification‎ can‎prevent‎sperm‎cryo-injuries.(6-9) While, it is shown that slow‎freezing‎and‎thawing‎is‎associated‎with‎sperm‎DNA‎ damage‎and‎apoptosis‎in‎human‎ejaculated‎spermatozoa,‎lit- tle‎is‎known‎about‎the‎effect‎of‎vitrification‎on‎induction‎of‎ human‎sperm‎DNA‎fragmentation.‎Cryopreservation‎of‎raw‎ or‎prepared‎semen‎has‎remained‎a‎matter‎of‎debate‎in‎the‎lit- erature.(2)‎Nawroth‎and‎colleagues‎reported‎that‎recovery‎rate‎ of‎motile‎spermatozoa‎as‎well‎as‎normal‎morphology‎after‎ vitrification‎was‎higher‎in‎native‎spermatozoa‎in‎comparison‎ to cryoprotectant used ones.(6)‎They‎also‎found‎that‎sperm‎re- covery‎rate‎and‎normal‎morphology‎will‎be‎higher‎after‎vit- rification‎in‎prepared‎spermatozoa‎compared‎to‎native‎group. (6)‎Recently,‎Satirapod‎and‎colleagues‎showed‎that‎the‎rate‎ of‎DNA‎fragmentation‎will‎be‎reduced‎in‎cryopreserved‎raw‎ semen‎with‎solid‎surface‎vitrification‎compared‎to‎standard‎ freezing‎method.(10) There‎ are‎ several‎ techniques‎ in‎ order‎ to‎ determine‎ sperm‎ DNA‎fragmentation.(11-13)‎Terminal‎deoxynucleotidyl‎trans- ferase‎dUTP‎nick‎end‎labeling‎(TUNEL)‎assay‎is‎a‎reliable‎ technique‎to‎evaluate‎double‎strand‎DNA‎fragmentation.(14) The‎main‎goal‎of‎this‎study‎was‎to‎evaluate‎the‎effect‎of‎vitri- fication‎of‎neat‎semen‎samples‎in‎both‎normal‎and‎abnormal‎ semen‎groups‎on‎the‎sperm‎parameters‎and‎DNA‎status‎using‎ TUNEL‎assay. MATERIALS AND METHODS Sampling and Spermatozoa Evaluation Ejaculates‎ were‎ obtained‎ from‎ men‎ aged‎ between‎ 30-50‎ years‎old‎(17‎normal‎and‎17‎abnormal‎semen‎samples)‎by‎ masturbation‎after‎48-hour‎of‎sexual‎abstinence.‎In‎normal‎ semen‎group,‎the‎inclusion‎criteria‎was‎infertility‎due‎to‎fe- male‎factor‎and‎in‎the‎infertile‎men‎the‎couples‎were‎infertile‎ due‎to‎male‎factor.‎After‎ liquefaction,‎semen‎analysis‎was‎ performed‎according‎to‎World‎Health‎Organization‎(WHO)‎ guidelines.(15)‎Sperm‎count‎and‎motility‎were‎assessed‎us- ing‎Neubauer‎chamber‎under‎the‎light‎microscope‎(×‎400).‎ Motility‎types‎were‎categorized‎into:‎progressive,‎non-pro- gressive,‎and‎immotile.‎The‎sperm‎viability‎was‎assessed‎us- ing‎eosin-nigrosin‎staining‎protocol.‎The‎dead‎spermatozoa‎ were‎stained‎red,‎while‎the‎live‎ones‎were‎unstained‎(Figure‎ 1).‎Also,‎sperm‎morphology‎was‎evaluated‎by‎Papanicolaou‎ staining‎procedure.‎At‎least,‎200‎spermatozoa‎were‎checked‎ under‎light‎microscope‎for‎head,‎neck‎and‎tail‎abnormalities.‎ Vitrification and Warming Vitrification‎method‎was‎according‎to‎previous‎reports‎with‎ some‎modifications.(16)‎The‎semen‎was‎loaded‎on‎copper‎cry- oloops‎of‎2.5‎mm‎diameter‎by‎dipping‎the‎loops‎in‎suspen- sion‎to‎obtain‎a‎thin‎film‎of‎8‎±‎2‎µL‎and‎the‎loaded‎loops‎ were‎plunged‎in‎the‎LN.‎After‎storage‎for‎7‎days,‎the‎samples‎ were‎warmed‎by‎plunging‎the‎loops‎into‎a‎tube‎containing‎ 2.5‎mL‎Ham's‎F10‎at‎37ºC.‎After‎warming‎of‎10‎loops‎in‎one‎ tube,‎the‎tube‎was‎placed‎in‎a‎CO2‎incubator‎for‎5-10‎min.‎ Then,‎the‎spermatozoa‎were‎centrifuged‎at‎300g‎for‎10‎min‎ and‎the‎resultant‎pellet‎was‎resuspended‎in‎100‎µL‎of‎Ham's‎ F10‎and‎processed‎for‎further‎evaluation. TUNEL Staining In‎Situ‎Cell‎Death‎Detection‎Kit‎(Roche‎Diagnostics‎GmbH.‎ Roche‎Applied‎Science.‎68298‎Mannheim,‎Germany)‎was‎ applied‎ for‎ TUNEL‎ assay.‎After‎ providing‎ the‎ smear,‎ the‎ slides‎were‎fixed‎in‎100%‎methanol‎solution‎for‎4‎min‎at‎ room‎temperature.‎Blocking‎was‎performed‎by‎putting‎the‎ slides‎in‎3%‎H2O2‎in‎methanol‎for‎10‎min‎in‎darkness.‎Before‎ and‎after‎blocking,‎the‎slides‎were‎washed‎with‎phosphate‎ buffered‎saline‎(PBS).‎For‎sperm‎permeabilization‎0.1%‎Tri- ton‎X-100‎in‎0.1%‎sodium‎citrate‎buffer‎was‎used‎(10‎min‎ 1467Vol. 11 | No. 02 | March- April 2014 |U R O LO G Y J O U R N A L Neat Semen Vitrification and Sperm Parameters | Khalili et al on‎ ice).‎ The‎ slides‎ were‎ incubated‎ with‎ TUNEL‎ reaction‎ mixture‎1-hour‎with‎high‎humidity‎at‎37˚C.‎After‎washing‎ with‎ PBS,‎ the‎ slides‎ were‎ incubated‎ with‎ convertor-probe‎ followed‎by‎incubation‎with‎3,3'-Diaminobenzidine‎(DAB)‎ (DAB,‎Roche,‎Mannheim,‎Germany)‎solution.‎Two‎hundred‎ sperm‎cells‎were‎analyzed‎under‎the‎light‎microscope‎at‎×‎ 1000.‎Abnormal‎spermatozoa‎had‎dark‎brown‎nuclear‎(Figure‎ 2).‎For‎positive‎controls‎0.1‎IU‎DNase‎(Hoffmann-La‎Roche‎ Diagnostics,‎Mannheim,‎Germany)‎was‎applied‎for‎15‎min‎at‎ 37˚C‎and‎the‎reaction‎mixture‎had‎no‎terminal‎deoxynucleoti- dyl‎transferase‎(TdT)‎for‎negative‎controls.‎ Statistical Analysis The‎data‎are‎shown‎as‎mean‎±‎SD.‎Sperm‎parameters‎before‎ and‎after‎vitrification‎was‎analyzed‎using‎paired‎t‎test.‎Linear‎ Pearson‎correlation‎test‎was‎applied‎to‎find‎out‎the‎correla- tion‎between‎the‎apoptosis‎and‎sperm‎parameters.‎The‎level‎ of‎statistical‎significance‎was‎set‎at‎P‎<‎.05. RESULTS The‎sperm‎cell‎count‎were‎120.70‎±‎68.14‎and‎15.15‎±‎2.58‎ (106/mL)‎in‎normal‎and‎abnormal‎semen‎groups,‎respectively.‎ Regarding‎sperm‎motility,‎56.11‎±‎10.45‎and‎28.11‎±‎8.15‎ were‎ progressive‎ motility‎ and‎ 67.58‎ ±‎ 10.01‎ and‎ 35.58‎ ±‎ 11.94‎were‎total‎motility,‎respectively.‎Sperm‎viability‎were‎ 78.47‎±‎9.38‎and‎50.58‎±‎15.15‎and‎sperm‎morphology‎were‎ 46.05‎±‎10.46‎and‎12.52‎±‎13.87,‎respectively. The‎data‎showed‎that‎sperm‎vitrification‎caused‎significant‎ decrease‎in‎sperm‎motility,‎viability‎and‎morphology‎in‎nor- mozoospermic‎samples‎(Table‎1).‎Also,‎vitrification‎was‎in- volved‎with‎significant‎increase‎in‎sperm‎DNA‎fragmentation‎ which‎was‎about‎8%‎in‎abnormal‎semen‎group.‎In‎addition,‎ there‎was‎significant‎reduction‎for‎all‎sperm‎parameters‎after‎ Table 1. Sperm parameters before and after vitrification in normozoospermic samples.* Sperm Parameters Before Vitrification After Vitrification P Count (×106/mL) 120.70 ± 68.14 89.00 ± 9.30 .134 Progressive motility (%) 56.11 ± 10.45 5.29 ± 5.05 .000 Total motility (%) 67.58 ± 10.01 8.64 ± 6.81 .000 Normal morphology (%) 46.05 ±10.46 37.00 ± 11.72 .024 Viability (%) 78.47 ± 9.38 11.05 ± 7.30 .000 TUNEL positive cells 16.41 ± 4.53 24.76 ± 5.03 .002 Key: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. *Data are shown as mean ± SD. Figure 1. Evaluation of human sperm viability using eosin- nigrosin staining; (a) unstained (white) alive spermatozoa, (b) stained (red) dead spermatozoa. Figure 2. Evaluating the sperm DNA fragmentation using TUNEL test. Dark brown cells are abnormal spermatozoa. Key: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. 1468 | Sexual Dysfunction And Infertility vitrification‎in‎abnormal‎semen‎group‎(Table‎2).‎ DNA‎fragmentation‎was‎11%‎higher‎in‎semen‎with‎abnormal‎ sperm‎parameters‎compared‎to‎baseline.‎There‎was‎negative‎ correlation‎between‎sperm‎DNA‎fragmentation‎and‎viability‎ in‎normozoospermic‎samples‎after‎vitrification‎(r = -0.6, P = .004).‎The‎negative‎correlation‎was‎only‎observed‎between‎ sperm‎DNA‎fragmentation‎and‎progressive‎motility‎after‎vit- rification‎in‎normozoospermic‎men‎(Table‎3).‎No‎significant‎ correlation‎was‎found‎between‎abnormal‎sperm‎DNA,‎viabil- ity‎and‎morphology‎after‎vitrification‎in‎semen‎with‎abnormal‎ sperm‎parameters‎(Table‎4).‎ DISCUSSION The‎data‎showed‎a‎significant‎decrease‎in‎sperm‎parameters‎ as‎well‎as‎significant‎increase‎in‎sperm‎DNA‎fragmentation‎ after‎vitrification‎in‎both‎groups‎of‎normal‎and‎abnormal‎se- men‎samples.‎Commonly,‎cryopreservation‎has‎negative‎im- pact‎on‎sperm‎motility‎and‎viability.‎Our‎data‎were‎similar‎to‎ others‎that‎cryopreservation‎caused‎decrease‎in‎sperm‎motil- ity‎and‎viability.(8,10)‎Satirapod‎and‎colleagues‎investigated‎ the‎efficacy‎of‎new‎vitrification‎method‎on‎normozoospermic‎ samples.‎They‎evaluated‎the‎role‎of‎raw‎semen‎solid‎surface‎ vitrification‎in‎comparison‎to‎rapid‎freezing‎method‎on‎sperm‎ parameters.‎Their‎data‎showed‎that‎sperm‎motility,‎viability,‎ morphology‎and‎DNA‎integrity‎were‎noticeably‎reduced‎after‎ vitrification.(10)‎In‎comparison‎to‎our‎study,‎their‎sperm‎recov- ery‎rate‎as‎well‎as‎DNA‎damage‎was‎higher.‎One‎probable‎ cause‎would‎be‎the‎method‎of‎sperm‎vitrification.‎Also‎they‎ used‎commercial‎cryoprotectant,‎while‎we‎used‎cryoprotect- ant‎free‎method.‎Nawroth‎and‎colleagues‎also‎reported‎re- duced‎sperm‎parameters‎after‎neat‎semen‎vitrification‎of‎nor- mal‎donors.‎The‎data‎showed‎that‎sperm‎recovery‎would‎be‎ much‎higher‎after‎swim‎up‎compared‎to‎native‎spermatozoa. (6)‎Formation‎of‎lethal‎intracellular‎ice‎crystal‎sand‎osmot- ic‎stress‎may‎be‎the‎main‎cause‎for‎reduction‎in‎sperm‎cell‎ motility‎and‎viability‎during‎cryopreservation.(17)‎Isachenko‎ and‎colleagues‎compared‎sperm‎motility‎after‎four‎different‎ cryoprotectant-free‎vitrification‎techniques‎and‎showed‎that‎ cryoloop‎method‎resulted‎in‎a‎lower‎sperm‎motility‎compared‎ to droplets, open pool straws and open straws.(18)‎Our‎data‎ also‎showed‎that‎vitrification‎impairs‎sperm‎normal‎morphol- ogy‎in‎normal‎and‎abnormal‎semen‎groups.‎The‎findings‎were‎ similar‎to‎other‎reports‎in‎terms‎of‎negative‎effects‎of‎cryo- preservation‎ on‎ normal‎ sperm‎ morphology.(1,10) It appears that‎the‎most‎probable‎reason‎for‎the‎effect‎of‎freezing‎on‎ sperm‎morphology‎is‎the‎formation‎of‎ice‎crystals‎outside‎the‎ sperm‎cell‎which‎can‎alter‎sperm‎architecture.(1) Generally,‎it‎is‎believed‎that‎normozoospermic‎semen‎sam- ples‎may‎be‎more‎resistant‎to‎cryo-injury‎compared‎to‎ab- normal‎ oligozoospermic‎ or‎ asthenozoospermic‎ specimens.‎ Table 2. Sperm parameters before and after vitrification in abnormal semen.* Sperm Parameters Before Vitrification After Vitrification P Count (×106/mL) 19.15 ± 2.58 17.82 ± 2.87 .806 Progressive motility (%) 42.11 ± 16.15 3.70 ± 2.35 .000 Total motility (%) 55.58 ± 18.94 7.88 ± 3.34 .000 Normal morphology (%) 18.52 ± 13.87 11.52 ± 9.57 .018 Viability (%) 60.58 ± 19.15 8.64 ± 3.66 .000 TUNEL positive cells 23.50 ± 8.31 34.29 ± 10.02 .000 Key: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. *Data are shown as mean ± SD. Table 3. Correlation between sperm DNA integrity and sperm parameters in normal semen before and after vitrification. Variables Progressive Motility Morphology Viability Count TUNEL positive spermatozoa (%) Before vitrification r = -0.23 r = -0.85 r = -0.03 r = 0.15 P = .35 P = .000 P = .89 P = .54 After vitrification r = -0.49 r = 0.45 r = -0.6 r = -0.53 P = .04 P = .06 P = .004 P = .02 Key: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. 1469Vol. 11 | No. 02 | March- April 2014 |U R O LO G Y J O U R N A L Neat Semen Vitrification and Sperm Parameters | Khalili et al Table 4. Correlation between apoptosis and sperm parameters in abnormal semen. Variables Progressive Motility Morphology Viability Count TUNEL positive spermatozoa (%) Before vitrification r = -0.73 r = 0.15 r = -0.67 r = 0.18 P = .001 P = .54 P = .003 P = .48 After vitrification r = -0.6 r = -0.37 r = -0.32 r = -0.12 P = .01 P = .13 P = .2 P = .62 Key: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. Our‎results‎showed‎that‎sperm‎parameters‎and‎DNA‎frag- mentation‎decreased‎at‎the‎same‎manner‎in‎both‎groups‎of‎ normal‎ and‎ abnormal‎ semen‎ groups,‎ which‎ is‎ in‎ conflict‎ with‎some‎reports.‎Donnelly‎and‎colleagues‎found‎that‎sper- matozoa‎from‎infertile‎men‎would‎be‎less‎resistant‎to‎cryo- injuries‎compared‎with‎spermatozoa‎of‎fertile‎men.(19)‎One‎ probable‎causes‎of‎this‎discrepancy‎may‎be‎method‎of‎cryo- preservation.‎They‎used‎rapid‎freezing‎method‎(freezing‎in‎ LN‎vapor),‎while‎our‎cryopreservation‎method‎was‎typical‎ vitrification.‎Also,‎we‎used‎no‎cryoprotectant‎in‎the‎freezing‎ method.‎Permeable‎cryoprotectant‎is‎used‎in‎slow‎freezing‎in‎ order‎to‎reduce‎cell‎shrinkage‎during‎cryopreservation.‎Using‎ permeable‎and‎non-permeable‎cryoprotectants‎in‎sperm‎cryo- preservation‎not‎only‎may‎have‎no‎beneficial‎effects,‎but‎also‎ can‎induce‎damage‎even‎at‎room‎temperature.(16) It has been reported‎that‎cryoprotectants‎have‎some‎cell‎toxicity‎such‎as‎ osmotic‎damage‎and‎chemical‎toxicity.(20) Regarding prob- able‎effect‎of‎cryoprotectants‎on‎sperm‎DNA,‎it‎is‎shown‎that‎ presence‎of‎cryoprotectants‎has‎no‎negative‎impact‎on‎sperm‎ DNA integrity.(16)‎Our‎data‎showed‎that‎vitrification‎can‎sig- nificantly‎increase‎sperm‎DNA‎fragmentation‎in‎both‎normal‎ and‎abnormal‎semen‎groups.‎The‎results‎were‎similar‎to‎the‎ Brazilian‎group.‎They‎found‎cryopreservation‎induced‎DNA‎ fragmentation‎in‎both‎oligozoospermic‎and‎normozoosper- mic‎samples.(21)‎and‎colleagues‎also‎showed‎that‎84.66%‎of‎ spermatozoa‎show‎undamaged‎DNA‎following‎vitrification/ warming‎in‎swim-up‎prepared‎normal‎specimens.(22)‎The‎ef- fect‎of‎cryopreservation‎on‎sperm‎DNA‎status‎has‎remained‎ controversial.‎Some‎investigators‎believe‎that‎cryopreserva- tion‎has‎no‎negative‎effect‎on‎sperm‎DNA‎status.(16,23) While, others‎ have‎ shown‎ that‎ the‎ cryopreservation‎ is‎ associated‎ with‎negative‎effect‎on‎sperm‎DNA‎integrity‎and‎chromatin‎ stability.(2,24)‎Oxidative‎stress‎may‎be‎one‎of‎the‎important‎ causes‎ of‎ increasing‎ sperm‎ DNA‎ fragmentation.(25)‎ Cryo- preservation‎may‎change‎the‎fluidity‎of‎sperm‎mitochondrial‎ membrane‎and‎consequently‎increase‎the‎potential‎of‎mito- chondrial‎membrane,‎finally‎reactive‎oxygen‎species‎(ROS)‎ will be produced and released.(2)‎We‎verified‎that‎semen‎sam- ples‎and‎non-sperm‎cells‎in‎seminal‎fluid‎are‎potential‎sources‎ of‎ROS‎production.‎Also,‎it‎is‎shown‎that‎presence‎of‎seminal‎ leukocytes‎is‎associated‎with‎more‎ROS‎production‎during‎ cooling‎to‎4˚C.(26)‎The‎thawing‎seems‎to‎have‎more‎impor- tant‎role‎in‎induction‎of‎DNA‎damage‎in‎sperm‎cells.‎It‎was‎ reported‎that‎the‎highest‎degree‎of‎sperm‎DNA‎fragmentation‎ will‎be‎occurred‎during‎the‎first‎4-hour‎of‎incubation‎after‎ thawing‎in‎fertile‎donors.(27) Another‎finding‎was‎the‎negative‎correlation‎between‎sperm‎ progressive‎motility‎and‎DNA‎fragmentation‎after‎vitrifica- tion‎in‎normozoospermic‎men.‎It‎was‎shown‎that‎there‎is‎a‎ negative‎relationship‎between‎sperm‎motility,‎vitality‎or‎con- centration‎and‎sperm‎DNA‎damage.(28,29)‎But,‎there‎was‎no‎ significant‎correlation‎between‎sperm‎morphology‎and‎DNA‎ integrity,‎ which‎ was‎ similar‎ to‎ other‎ findings.(19)‎ It‎ seems‎ that‎the‎sperm‎morphological‎feature‎is‎not‎representative‎of‎ sperm‎DNA‎quality.‎Cryopreservation‎of‎raw‎or‎prepared‎se- men‎has‎remained‎matter‎of‎debate‎in‎the‎literature.(2) It is believed‎ that‎seminal‎plasma‎contains‎natural‎antioxidants‎ which‎ can‎ protect‎ spermatozoa‎ from‎ cyro-injuries‎ during‎ cryopreservation‎and‎these‎seminal‎plasma‎antioxidants‎will‎ be‎eliminated‎with‎sperm‎preparation‎methods.‎Neat‎semen‎ cryopreservation‎would‎be‎rapid‎and‎cost-effective‎as‎well.‎ In‎this‎study‎we‎cryopreserved‎normal‎and‎abnormal‎raw‎se- men.‎Maybe,‎ROS‎production‎by‎non-sperm‎cells‎in‎seminal‎ plasma‎is‎higher‎than‎seminal‎plasma‎antioxidants‎capacity,‎ especially‎in‎sub‎normal‎specimens.‎ CONCLUSION Vitrification‎of‎human‎neat‎semen‎can‎impair‎vital‎sperm‎pa- rameters‎of‎motility,‎viability,‎morphology‎as‎well‎as‎DNA‎ integrity.‎It‎might‎be‎better‎to‎vitrify‎the‎processed‎semen,‎ especially‎for‎cases‎with‎male‎factor‎infertility. CONFLICT OF INTEREST None declared. 1470 | Sexual Dysfunction And Infertility REFERENCES 1. Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Ef- fects of cryopreservation on sperm parameters and ultrastruc- tural morphology of human spermatozoa. J Assist Reproduc Genet. 2008;25:403-11. 2. Said TM, Gaglani A, Agarwal A. Implication of apoptosis in sperm cryoinjury. Reprod Biomed Online. 2010;21:456-62. 3. Di Santo M, Tarozzi N, Nadalini M, Borini A. Human Sperm Cryo- preservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART. Adv Urol. 2012;2012:3. 4. Al-Hasani S, Ozmen B, Koutlaki N, Schoepper B, Diedrich K, Schultze- Mosgau A. Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? Reprod Biomed Online. 2007;14:288-93. 5. Fahy GM. The relevance of cryoprotectant “toxicity” to cryobiology. Cryobiology. 1986;23:1-13. 6. Nawroth F, Isachenko V, Dessole S, et al. Vitrification of human sper- matozoa without cryoprotectants. Cryo Letters. 2002;23:93-102. 7. Isachenko E, Isachenko V, Katkov II, Dessole S, Nawroth F. Vitrifica- tion of mammalian spermatozoa in the absence of cryoprotectants: from past practical difficulties to present success. Reprod Biomed Online. 2003;6:191-200. 8. Isachenko E, Isachenko V, Weiss J, et al. Acrosomal status and mi- tochondrial activity of human spermatozoa vitrified with sucrose. Reproduction. 2008;136:167-73. 9. Isachenko V, Maettner R, Petrunkina A, et al. Vitrification of human ICSI/IVF spermatozoa without cryoprotectants: new capillary tech- nology. J Androl. 2012;33:462-8. 10. Satirapod C, Treetampinich C, Weerakiet S, Wongkularb A, Rattana- siri S, Choktanasiri W. Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing meth- od: on motility, morphology, vitality and DNA integrity. Andrologia. 2012;44 Suppl 1:786-90. 11. Khalili MA, Aghaie-Maybodi F, Anvari M, Talebi AR. Sperm nuclear DNA in ejaculates of fertile and infertile men: correlation with se- men parameters. Urol J. 2006;3:154-9. 12. Nabi A, Khalili MA, Halvaei I, Roodbari F. Prolonged incubation of processed human spermatozoa will increase DNA fragmentation. Andrologia. (Article first published online : 12 MAR 2013, DOI: 10.1111/and.12088. 13. Halvaei I, Sadeghipour Roodsari HR, Naghibi Harat Z. Acute Effects of Ruta graveolens L. on Sperm Parameters and DNA Integrity in Rats. J Reprod Infertil. 2012;13:33-8. 14. Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. 1992;119:493-501. 15. WHO. WHO laboratory manual for the Examination and processing of human semen. 5 ed: Cambridge University Press; 2010. 16. Isachenko V, Isachenko E, Katkov II, et al. Cryoprotectant-free cryo- preservation of human spermatozoa by vitrification and freezing in vapor: effect on motility, DNA integrity, and fertilization ability. Biol Reprod. 2004;71:1167-73. 17. Muldrew K, McGann LE. Mechanisms of intracellular ice formation. Biophys J. 1990;57:525-32. 18. Isachenko V, Isachenko E, Montag M, et al. Clean technique for cryoprotectant-free vitrification of human spermatozoa. Reprod Biomed Online. 2005;10:350-4. 19. Donnelly ET, Steele EK, McClure N, Lewis SE. Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation. Hum Reprod. 2001;16:1191-9. 20. Katkov II, Katkova N, Critser JK, Mazur P. Mouse spermatozoa in high concentrations of glycerol: chemical toxicity vs osmotic shock at normal and reduced oxygen concentrations. Cryobiology. 1998;37:325-38. 21. de Paula TS, Bertolla RP, Spaine DM, Cunha MA, Schor N, Cedenho AP. Effect of cryopreservation on sperm apoptotic deoxyribonucle- ic acid fragmentation in patients with oligozoospermia. Fertil Steril. 2006;86:597-600. 22. Isachenko E, Isachenko V, Katkov II, et al. DNA integrity and motility of human spermatozoa after standard slow freezing versus cryo- protectant‐free vitrification. Hum Reprod. 2004;19:932-9. 23. Duty S, Singh N, Ryan L, et al. Reliability of the comet assay in cryo- preserved human sperm. Hum Reprod. 2002;17:1274-80. 24. Hammadeh M, Askari A, Georg T, Rosenbaum P, Schmidt W. Effect of freeze-thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl. 1999;22:155-62. 25. Thomson L, Fleming S, Aitken R, De Iuliis G, Zieschang J-A, Clark A. Cryopreservation-induced human sperm DNA damage is predomi- nantly mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24:2061-70. 26. Wang AW, Zhang H, Ikemoto I, Anderson DJ, Loughlin KR. Reactive oxygen species generation by seminal cells during cryopreserva- tion. Urology. 1997;49:921-5. 27. Gosálvez J, Cortés-Gutierez E, López-Fernández C, Fernández JL, Caballero P, Nuñez R. Sperm deoxyribonucleic acid fragmentation dynamics in fertile donors. Fertil Steril. 2009;92:170-3. 28. Shen HM, Dai J, Chia SE, Lim A, Ong CN. Detection of apoptotic al- terations in sperm in subfertile patients and their correlations with sperm quality. Hum Reprod. 2002;17:1266-73. 29. Zhang HB, Lu SM, Ma CY, Wang L, Li X, Chen ZJ. Early apoptotic changes in human spermatozoa and their relationships with con- ventional semen parameters and sperm DNA fragmentation. Asian J Androl. 2008;10:227-35. 1471Vol. 11 | No. 02 | March- April 2014 |U R O LO G Y J O U R N A L Vitrification‎has‎brought‎about‎important‎changes‎in‎cryopreservation‎and‎human‎fer-tility‎preservation.‎Easiness‎and‎speed‎and‎no‎need‎for‎costly‎freezing‎technologies‎are‎reasons‎for‎its‎rapid‎development.‎Vitrification‎is‎the‎solidification‎of‎a‎liquid‎ without‎crystallization.‎As‎cooling‎continues,‎however,‎the‎molecular‎waves‎in‎the‎liquid‎per- meating‎the‎tissue‎decline.‎Finally,‎an‎"arrested‎liquid"‎state‎known‎as‎a‎glass‎is‎attained.‎Vit- rification‎has‎been‎demonstrated‎to‎afford‎higher‎preservation‎for‎a‎number‎of‎cells,‎including‎ monocytes,‎ova‎and‎early‎embryos‎and‎pancreatic‎islets.(1) There‎are‎a‎number‎of‎major‎contests‎for‎performing‎of‎vitrification‎for‎tissue‎engineered‎ medical‎products.‎Without‎adhering‎to‎these‎standards,‎certainly‎the‎process‎of‎vitrification‎ will‎fail.‎The‎first‎one‎is‎vitreous‎state.‎There‎is‎no‎explanation‎about‎vitreous‎state‎in‎this‎ study.‎Stability‎of‎the‎vitreous‎state‎is‎critical‎for‎the‎maintenance‎of‎vitrified‎tissue‎integrity‎ and‎viability.‎In‎present‎study‎the‎method‎of‎vitrification‎has‎not‎been‎explained‎in‎details‎and‎ it‎seems‎most‎of‎standards‎for‎vitrification‎have‎not‎been‎considered.‎Vitrification‎methods‎to‎ preservation‎have‎some‎of‎the‎limitations‎associated‎with‎conventional‎freezing‎methods.(2) First,‎both‎methods‎entail‎low‎temperature‎storage‎and‎transportation‎conditions.‎Neither‎can‎ be‎stored‎above‎their‎glass‎transition‎temperature‎for‎long‎without‎significant‎risk‎of‎product‎ damage‎due‎to‎inherent‎instabilities‎resulting‎to‎ice‎formation‎and‎growth.‎Both‎methods‎use‎ cryoprotectants‎with‎their‎associated‎problems‎and‎necessitate‎experienced‎technical‎support‎ during‎rewarming‎and‎cryoprotectant‎elution‎phases.‎The‎very‎high‎concentrations‎of‎cryopro- tectants‎needed‎to‎facilitate‎vitrification‎are‎potentially‎toxic‎since‎the‎cells‎may‎be‎exposed‎to‎ these‎high‎concentrations‎at‎higher‎temperatures‎than‎in‎freezing‎methods‎of‎cryopreservation.‎ Cryoprotectants‎can‎kill‎cells‎by‎direct‎chemical‎toxicity,‎or‎indirectly‎by‎osmotically-induced‎ stresses‎during‎suboptimal‎addition‎or‎removal.(3)‎Upon‎complete‎achievement‎of‎warming,‎ the‎cells‎should‎not‎be‎exposed‎to‎temperatures‎above‎0oC‎for‎more‎than‎a‎few‎minutes‎before‎ the‎glass-forming‎cryoprotectants‎are‎removed.‎It‎is‎possible‎to‎employ‎vitrified‎products‎in‎ highly‎controlled‎environments,‎such‎as‎a‎commercial‎manufacturing‎facility‎or‎an‎operating‎ theater,‎but‎not‎in‎an‎outpatient‎office.‎There‎isn’t‎any‎data‎about‎above‎mentioned‎points‎in‎ this study.(4)‎Another‎issue‎is‎heat‎transfer.‎Heat‎transfer‎issues‎are‎the‎primary‎problem‎for‎ scaling‎up‎the‎successes‎in‎somewhat‎small‎tissue‎specimens‎to‎larger‎tissues‎and‎organs.‎The‎ limits‎of‎heat‎and‎mass‎transfer‎in‎bulky‎systems‎result‎in‎non-uniform‎cooling‎and‎leads‎to‎ stresses‎that‎might‎begin‎cracking.‎In‎fact,‎the‎higher‎cooling‎rates‎that‎facilitate‎vitrification‎ will‎typically‎lead‎to‎higher‎mechanical‎stresses.(5)‎In‎present‎study‎there‎is‎no‎information‎on‎ the‎used‎material‎properties‎of‎vitreous‎aqueous‎solutions.‎Material‎properties‎such‎as‎thermal‎ conductivity‎and‎fracture‎strength‎of‎vitreous‎aqueous‎solutions‎have‎many‎connections‎with‎ their‎inorganic‎analogues‎that‎happen‎at‎normal‎temperatures.‎Any‎material‎that‎is‎unrestricted‎ will‎undergo‎a‎change‎in‎size‎(thermal‎strain)‎when‎subjected‎to‎a‎change‎in‎temperature.‎ Additional‎important‎issue‎that‎has‎not‎been‎addressed,‎is‎the‎stresses‎that‎arise‎to‎billet‎the‎ differential‎shrinkage.‎Thermal‎stress‎can‎definitely‎reach‎the‎produced‎strength‎of‎the‎frozen‎ tissue‎resulting‎in‎plastic‎deformations‎or‎fractures.(6)‎One‎more‎major‎obstacle‎for‎performing‎ Editorial comment on: Vitrification of Neat Se- men Alters Sperm Parameters and DNA Integrity Mohammad Reza Safarinejad M.D Clinical Center for Urological Disease Diagnosis and Private Clinic Special- ized in Urological and Andrological Genetics, Tehran, Iran. E-mail: info@safarinejad.com 1472 | of‎vitrification‎is‎the‎technique‎used‎for‎warming.‎This‎issue‎also‎has‎been‎ignored‎in‎present‎ study.‎The‎warming‎technique‎should‎be‎highly‎effective‎to‎prevent‎devitrification‎and‎ice‎ growth by recrystallization. The‎rational‎for‎vitrification‎of‎neat‎semen‎has‎not‎been‎mentioned.‎What‎are‎the‎advantages‎ of‎vitrification‎of‎semen‎instead‎of‎sperm?‎Is‎there‎any‎scientific‎background‎for‎this‎proce- dure?‎For‎vitrification,‎it‎is‎recommended‎that,‎even‎the‎plasma‎of‎sperm‎should‎be‎removed.‎ For‎vitrification‎the‎sperm‎plasma‎is‎removed,‎it‎means‎that‎by‎using‎this‎technique‎many‎ infecting‎agents‎such‎as‎HIV,‎hepatitis‎and‎other‎viruses‎will‎be‎removed‎from‎the‎sperm,‎and‎ therefore‎these‎infectious‎microorganism‎cannot‎be‎transmitted‎via‎sperm.‎Hence‎HIV+‎men‎ will‎have‎the‎chance‎to‎father‎children‎without‎the‎risk‎of‎passing‎infectious‎organisms‎to‎baby‎ and‎mother.‎After‎separation‎of‎plasma‎from‎the‎sperm,‎the‎vitrified‎sperm‎should‎be‎stored‎in‎ an‎ultra-cold‎deep‎freeze‎at‎-86ºC‎environment.‎This‎method‎has‎several‎advantages‎compared‎ to‎other‎methods,‎first‎the‎motility‎of‎rethawed‎sperm‎increases‎significantly‎(75%‎using‎this‎ method‎vs.‎31%‎using‎conventional‎methods)‎second‎a‎higher‎number‎of‎viable‎sperm‎can‎be‎ achieved‎and‎this‎can‎result‎in‎higher‎chance‎of‎fertilization‎in‎ARTs,‎such‎as‎IVF‎and‎ICSI.(7) However,‎two‎decades‎past‎the‎first‎live-birth‎from‎vitrified‎embryos,‎there‎are‎still‎some‎ uncertainties‎on‎the‎safety‎of‎these‎techniques‎and‎its‎possible‎toxic‎effects‎on‎the‎health‎of‎ children‎born‎from‎vitrified‎embryos‎or‎oocytes.‎There‎is‎fear‎that‎use‎of‎high‎concentrations‎ of‎cryoprotectants‎may‎result‎in‎genetic‎or‎epigenetic‎abnormalities‎with‎ensuing‎inborn‎mal- formations.‎Therefore,‎there‎is‎no‎agreement‎or‎scientific‎recommendations‎for‎the‎replace- ment‎of‎slow‎freezing‎method‎with‎vitrification‎universally. The‎techniques‎for‎performing‎vitrification‎are‎evolving.‎Recently‎vitrification‎of‎metaphase‎ II‎oocytes‎has‎been‎described‎to‎hold‎ability‎for‎oocyte‎preservation,‎which‎can‎be‎vital‎in‎ countries‎where‎a‎limited‎number‎of‎oocytes‎can‎be‎inseminated‎and‎embryo‎cryopreservation‎ is‎illegal,‎as‎well‎as‎in‎oocyte‎donation‎and‎fertility‎preservation‎prior‎to‎cancer‎treatment.(8) The‎two‎most‎commonly‎used‎tests‎to‎determine‎sperm‎DNA‎damage‎are‎the‎TUNEL‎as- say‎and‎the‎sperm‎chromatin‎structure‎assay‎(SCSA).(9)‎the‎TUNEL‎assay‎has‎never‎been‎ adjusted‎for‎use‎with‎human‎spermatozoa‎and‎lower‎normal‎threshold‎values‎have‎not‎been‎ obviously‎recognized.‎DNA‎testing‎by‎SCSA‎has‎been‎widely‎standardized.‎TUNEL‎test‎has‎ not‎been‎standardized‎to‎the‎same‎level‎as‎SCSA.‎TUNEL‎assay‎cannot‎selectively‎differenti- ate‎clinically‎significant‎DNA‎fragmentation‎from‎clinically‎insignificant‎fragmentation.‎The‎ assay‎also‎cannot‎differentiate‎normal‎DNA‎grooves‎from‎pathologic‎grooves.‎Moreover,‎the‎ TUNEL‎test‎does‎not‎give‎any‎information‎concerning‎the‎particular‎genes‎that‎may‎be‎af- fected‎by‎DNA‎fragmentation.‎This‎assay‎can‎only‎determine‎the‎amount‎of‎DNA‎fragmenta- tion‎that‎ensues,‎with‎the‎hypothesis‎that‎higher‎levels‎of‎DNA‎fragmentation‎are‎pathologic. (10)‎Nowadays,‎the‎only‎reliable‎test‎to‎determine‎sperm‎DNA‎fragmentation‎is‎SCSA.‎This‎ test‎has‎validated‎clinical‎reference‎range‎and‎criteria‎to‎interpret‎the‎yielded‎results‎precisely.‎ Using‎the‎SCSA‎test‎one‎can‎test‎5,000‎individual‎sperm‎with‎a‎high-precision‎flow‎cytometer.‎ To‎interpret‎the‎results‎of‎SCSA‎test‎DNA‎fragmentation‎index‎(DFI)‎is‎used,‎which‎represents‎ the‎population‎of‎cells‎with‎DNA‎damage.(11,12) Finally‎a‎major‎limitation‎of‎present‎study‎is‎absence‎of‎pictures‎both‎from‎TUNEL‎results‎ and‎vitrified‎sperms. Sexual Dysfunction and Infertility 1473Vol. 11 | No. 02 | March- April 2014 |U R O LO G Y J O U R N A L REFERENCES 1. Arav A, Natan Y. Vitrification of oocytes: from basic science to clinical application. Adv Exp Med Biol. 2013;761:69-83. 2. Aerts JM, De Clercq JB, Andries S, Leroy JL, Van Aelst S, Bols PE. Fol- licle survival and growth to antral stages in short-term murine ovar- ian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing. Cryobiology. 2008;57:163-9. 3. Merino O, Aguagüiña WE, Esponda P, et al. Protective effect of bu- tylated hydroxytoluene on sperm function in human spermatozoa cryopreserved by vitrification technique. Andrologia. 2014 Feb 24. doi: 10.1111/and.12246. [Epub ahead of print] 4. Imaizumi K, Nishishita N, Muramatsu M, et al. A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol. PLoS One. 2014;9:e88696. 5. Steif PS, Palastro M, Wan CR, Baicu S, Taylor MJ, Rabin Y. Cryomacros- copy of vitrification, Part II: Experimental observations and analysis of fracture formation in vitrified VS55 and DP6. Cell Preserv Technol. 2005;3:184-200. 6. Rabin Y, Podbilewicz B. Temperature-controlled microscopy for im- aging of living cells: apparatus, thermal analysis, and temperature dependency of embryonic elongation in Caenorhabditis elegans. J Microsc. 2000;199:214-23. 7. Steif PS, Palastro MC, Rabin Y. The Effect of Temperature Gradients on Stress Development During Cryopreservation via Vitrification. Cell Preserv Technol. 2007;5:104-15. 8. Baicu S, Taylor MJ, Chen Z, Rabin Y. Cryopreservation of carotid ar- tery segments via vitrification subject to marginal thermal condi- tions: correlation of freezing visualization with functional recovery. Cryobiology. 2008;57:1-8. 9. Zini A, Boman JM, Belzile E, et al. Sperm DNA damage is associated with an increased risk of pregnancy loss after IVF and ICSI: system- atic review and meta-analysis. Hum Reprod. 2008;23:2663-8. 10. Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influ- enced by DNA compaction and cell vitality: development of an im- proved methodology. Int J Androl. 2011;34:2-13. 11. Safarinejad MR. Sperm DNA damage and semen quality impair- ment after treatment with selective serotonin reuptake inhibitors detected using semen analysis and sperm chromatin structure as- say. J Urol. 2008;180:2124-8. 12. Safarinejad MR. Sperm Chromatin Structure Assay Analysis of Iranian Mustard Gas Casualties: A Long-Term Outlook. Curr Urol. 2010;4:71-80. Neat Semen Vitrification and Sperm Parameters | Khalili et al