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