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EDITORIAL The azoospermic male: current knowledge and future perspectives Sandro C. Esteves,I* Ashok AgarwalII* IANDROFERT,ClıńicadeAndrologiaeReproduçaõHumanaeCentrodeRefereˆnciaparaReproduçaõMasculina,Campinas,SaõPaulo,Brazil.IICenterfor ReproductiveMedicine,ClevelandClinicFoundation,Cleveland,Ohio,USA.*GuestEditors. This special issue is fully dedicated to the topic of azoospermia and contains the seminal work of renowned scientists and clinicians from seven countries on three continents. In seventeen chapters, a comprehensive reviewoftheepidemiology,genetics,physiopathology,diagnosis,andmanagementofazoospermiaaddresses our current knowledge on the topic. The clinical results of assisted reproductive techniques applied to this categoryofmaleinfertilityandthehealthofoffspringoriginatingfromsuchfathersarecriticallyanalyzed.In addition,thechallengesandthefuturebiotechnologicalperspectivesforthetreatmentofazoospermicmales seekingfertility arediscussed. EstevesSC,AgarwalA.Theazoospermicmale:currentknowledgeandfutureperspectives.Clinics.2013;68(S1):1-4. E-mail:[email protected] Tel.:+55193295-8877 Twomajorbreakthroughsrevolutionizedthefieldofmale invited leading, internationally recognized scientists and infertility in the last three decades. The first was the clinicians from the various sub-specialties to compile a development of intracytoplasmic sperm injection (ICSI) for collection of high-quality and comprehensive reviews the treatment of male factor infertility, and the second was highlighting the most current advances and contentious application of ICSI to azoospermic males, with the demon- issuesinazoospermia.Ouraimistoprovidereaderswitha stration that spermatozoa derived from either the epididy- thoughtful and wide-ranging review of the epidemiology, mis or the testis were capable of normal fertilization and genetics, physiopathology, diagnosis, and management of pregnancy. Azoospermia, defined as the complete absence azoospermia. The text is the first of its type and represents of spermatozoa in the ejaculate, invariably results in aninvaluabletoolforbothbasicscientistswithaninterestin infertility but does not necessarily imply sterility. In fact, sperm biology and clinicians (urologists, gynecologists, azoospermia has been recognized as one of the most reproductiveendocrinologists, andembryologists)working in the field of infertility. The selection of topics demon- intriguing topics in male infertility. Due to the true nature strates the exciting breadth of this category of male ofresearchinvolvingtheclassicaldisciplinesofphysiology, infertility and the opportunity that research in this area biochemistry and molecular biology, a rapid rise in the holdsforbothunderstandingandimprovingthereproduc- volume of scientific knowledge regarding azoospermia has tive healthofazoospermicmales. been obtained. Invariably, this has led to a better under- This Special Issue commences with provocative insights standing of the multi-faceted aspects of azoospermia. into the genetic and epigenetic paternal contribution to the However, there is still relatively little data within the human embryo (1). Dada and colleagues from New Delhi literature supporting common clinical practices. The repro- presentthecurrentknowledgeontheroleofspermatozoaas ductive potential of azoospermic males with different highly specialized cells with the purpose of not only etiologies is unclear; furthermore, the association of an delivering competent paternal DNA to the oocyte but also increased risk of birth defects and potential iatrogenic providing a robust epigenetic contribution to embryogen- transmissionofgeneticabnormalitieswithICSIusingsperm esis.Thepaternalepigeneticcontributiontoembryogenesis retrieved fromthesepatients is stillunder debate. requires that both the sperm DNA and the chromatin Thescopeofazoospermiarelated-infertilitynowcoversa structure as a whole contain layers of regulatory elements widespectrum,includinggeneticstudies,hormonalcontrol, that are sufficient to drive genes towards activation or microsurgical and medical therapy, assisted reproduction silencing upon delivery to the egg. Changes in the techniques, and innovative stem cell research that aims to epigenome are now known to affect gene expression, and create artificial gametes. In this special issue of Clinics, we several genes participating in spermatogenesis have been demonstrated tobe epigenetically regulated. The second article by Marcello Cocuzza and colleagues Copyright (cid:2) 2013CLINICS–ThisisanOpenAccessarticledistributedunder fromtheUniversityofSaõPauloisacomprehensivereview thetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http:// of the epidemiology and etiology of azoospermia. creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non- commercial use, distribution, and reproduction in any medium, provided the According to these authors, azoospermia is identified in originalworkisproperlycited. approximately 1% of all men and 10% to 15% of infertile DOI:10.6061/clinics/2013(Sup01)01 males (2). With a population of approximately 3 billion 1 Editorialforthespecialissueonazoospermia CLINICS2013;68(S1):1-4 EstevesSandAgarwalA people at reproductive age, a gross estimate indicates that duct. OA is considered to be one of the most favorable approximately10millionmenworldwideareazoospermic. prognostic conditions for male infertility because sperma- The authors explore several conditions that may lead to togenesis is not disrupted, unlike in NOA. Drs. Baker and azoospermia and didactically divide them into pre-testicu- Sabanegh from the Cleveland Clinic discuss the current lar, post-testicular, and testicular causes. Despite the indications, techniques and results of reconstructive advances in the diagnostic tools that identify men with procedures in OA (6). These authors highlight genetic-originazoospermia,manymenarestillclassifiedas the refinements in microsurgery that have optimized the having idiopathic azoospermia because the specific etiolo- success of reconstructive procedures; additionally, the gical factor remains unidentifiable. Therefore, determining authors indicate that the use of optical magnification is the etiology of azoospermia remains one of the main now the gold standard for vasal reconstruction. While the challenges inthis field. results of reconstructive procedures are excellent follow- Drs. Gudeloglu and Parekattil from the United States ing vasectomies, other complex repairs may be required, follow the theme by highlighting the importance of the especially in other etiological categories of OA. Despite clinical exam in the investigation of the azoospermic male. being highly successful, microsurgical reconstruction may The authors detailed review defines why and how the not be indicated in all men with OA, such as in patients clinicalevaluationshouldbeundertaken.Theyalsodiscuss with congenital bilateral absence of vas deferens (CBAVD) theusefulnessandlimitationsoftestisbiopsiesandimaging and certain cases of post-infectious obstructions or failed studiesinthecontextofazoospermiaandtheimportanceof vasectomy reversals. In such cases, sperm retrieval can be geneticcounselingbeforeusingthespermatozoafrommen performed for use with ICSI. with nonobstructive azoospermia (NOA) for assisted Nonobstructive azoospermia, on the other hand, poses a reproductivetechniques(3).Dr.Azizprovidesalaboratory differentchallenge.Fromthemanagementstandpoint,men perspective from the United Kingdom by cleverly defining with NOA are the most difficult to treat, and extensive azoospermia as a descriptive term for ejaculates that lack debateexistsonthebenefitofinterventionforthiscategory spermatozoa without implying a specific underlying cause of male infertility. Various conditions may cause NOA, (4).Theauthorstressesthatpropertechniquesareneededto including genetic and congenital abnormalities, post-infec- reduce the amount of analytical error and enhance sperm tious issues, exposure to gonadotoxins, medications, var- count precision when evaluating semen specimens. The icocele,trauma,endocrinedisorders,andidiopathiccauses. correctassessmentofaninitiallyazoospermicsemenspeci- This Special Issue contains three articles that critically men should be followed by an examination of the pelleted explore the role of medical and surgical therapy in sementoexcludecryptozoospermia,whichisdefinedbythe nonobstructive azoospermia. presence of a very small number of live sperm in a Renato Fraietta and colleagues from the Federal centrifuged pellet. An accurate assessment of very low University of Saõ Paulo provide a timely review on spermcountsaimstoavoidlabelingseverelyoligozoosper- hypogonadotropic hypogonadism (HH), which is a failure micmenasazoospermic,whichisparticularlyimportantin ofspermatogenesisduetoalackofappropriatestimulation the current era of assisted reproduction technology. Dr. bygonadotropins(7).Thiscategoryofpatientsincludesnot Azizs chapter provides insightful information on the onlycongenitalformsofHHbutalsoasubsetofmenwhose seminal plasma biomarkers that may aid in determining spermatogenic potential has been suppressed by excess thecausesofazoospermia.Laboratoryseminologyisclearly androgens or steroids. These patients benefit from specific movingfromtheassessmentofconventionalsemenprofiles hormonal therapy and often show remarkable recovery of intotheassessmentofspermfunction.Thisstrategyislikely spermatogenic function with exogenously administered toaidintheunderstandingoftheunderlyingpathophysiol- gonadotropins or gonadotropin releasing hormone. ogyofmaleinfertility,andnoninvasivebiomarkersmaybe Unfortunately, not all men with NOA have HH. In fact, useful in discriminating NOA cases from obstructive the larger category of NOA consists of men with intrinsic azoospermia (OA). testicular impairment in which empirical medical therapy This Special Issue contains an authoritative article that showslittlebenefit,asnotedbyDr.RajeevKumarfromthe provide the current knowledge on the genetic aspects of All India Institute of Medical Sciences in New Delhi (8). azoospermia and the testing available for clinical use. Dr. However, due to the developments in ART, a renewed Hamada and his co-authors from the Cleveland Clinic interest in the role of interventions in this subset of NOA provide an immense summary of results on the genetic patients has developed. The author expertly discusses the aspectsofmaleinfertilityandimplicationoftheseresultson role of medical therapy in these men to improve the the diagnosis and treatment of azoospermic males (5). quantity and quality of sperm that can be eventually Molecular biology genetic testing involving the Y-chromo- retrieved from their ejaculates or from their testes for use some can now correctly identify azoospermic men mis- in ICSI. In this sense, gonadotropins, aromatase inhibitors diagnosed as having idiopathic infertility. Moreover, Y- andnon-steroidalantiestrogens showpromise inachieving chromosome testing is of prognostic value for sperm thisendpoint.Lastly,Dr.KubilayIncifromTurkeyprovides retrieval in NOA. The authors also present practical a critical appraisal on the role of varicocele repair for recommendations for testing,andtheydiscuss thepossible men with NOA (9). His authoritative review discusses implications of using spermatozoa from men with genetic thepathophysiologyofvaricocele-relatedinfertility,andthe abnormalities forassisted conception. discussion is supported by his own experience on the Intheclinicalsetting,azoospermicpatientsarediagnosed impact of varicocele repair in azoospermic patients. From as having obstructive or nonobstructive azoospermia. the limited published data, it has been suggested that Obstructiveazoospermiahasbeenattributedtoamechanical varicocele repair may not only allow small quantities of blockage that can occur anywhere along the reproductive sperm to appear in the ejaculate but also may enhance the tract,includingthevasdeferens,epididymis,andejaculatory chancesofretrievingspermfromthetestisofthesepatients. 2 CLINICS2013;68(S1):1-4 Editorialforthespecialissueonazoospermia EstevesSandAgarwalA Even a minimal restoration of sperm production facilitates techniques are described for optimizing the chances of sperminjection procedures. harvesting spermatozoa from epididymal fluid and testi- Currently, ART is the only option for most men with cular tissue of azoospermic men. The concept of cryopre- azoospermia-related infertility to have their biological off- servation may also be used in association with sperm spring.SuccesshasbeenachievedwithICSIinbothobstructive retrieval procedures. Some centers prefer to retrieve and andnonobstructiveazoospermia,andtheuseofnon-ejaculated intentionally cryopreserve sperm for future use. This spermcoupledwithICSIhasbecomeaworldwideestablished strategy offers the advantage of avoiding ovarian stimula- procedure.Surgicalmethodshavebeendevelopedtoretrieve tionwhennospermisobtainedfromtesticularspecimens.If spermatozoa from the epididymides and testes. After sperm spermisretrievedandfrozen,itcanbethawedatanytime, retrieval, ICSI is used rather than standard IVF because ICSI therebyavoidingtheneedtoorganizetwooperations(oocyte hasbeenshowntoresultinasignificantlyhigherfertilization and sperm retrieval) on the same day. Additionally, rate.AsectionofthisSpecialIssuecomprisedofsevenarticles cryopreservation may spare unused specimens that would is fully dedicated to the use of assisted conception in bedischargedafterICSI,whichmaybeusefulifthetreatment azoospermia-related infertility. The authors of these selected cycle does not result in a pregnancy. Therefore, future ICSI titles have extensive publication records and more than a attempts could be conducted without repeated surgical decadeofclinicaland/orlaboratoryexperienceinthemanage- retrievals. In most cases of epididymal retrievals, motile ment of azoospermic males using assisted conception. In the sperm will be available after thawing, and ICSI outcomes firstarticleofthissection,theauthorspreparedacomprehen- using fresh motile or frozen-thawed epididymal sperm do sivesummaryofthecurrentmethodsforspermretrievaland not seem to differ. Cryopreservation of testicular sperm is criticallyanalyzedtheadvantages anddisadvantagesofeach also advisable, especially for men with NOA who often method (10). The authors note that the sperm retrieval (SR) requiremultipleICSIattemptstoconceivebutmaynothave methodofchoiceisoftenbasedonthetypeofazoospermiaand anadequatenumberofspermavailableforrepeatedretrieval theattendingsurgeon’spreferences.However,SRshouldaim attempts. These important aspects are discussed by Dr. to both minimize damage to the reproductive tract, thereby Gangrade from Orlando, who also presents laboratory preserving the chance of repeated retrieval attempts, and to protocols for the cryopreservation of epididymal and obtainanadequatenumberofgoodqualityspermthatcanbe testicular sperm and discusses the reproductive outcomes immediately used for ICSI or alternatively cryopreserved for ofusingfrozen-thawedgametesforsperminjections(14). future ICSI attempts.Following this theme, two articles from The closing articles of the section dedicated to assisted Brazilian groups discuss the key elements for the success of reproduction are authored by the guest editors and Drs. spermretrievalinobstructiveandnonobstructiveazoospermia Veerle and colleagues from the Centre of Reproductive (11,12). Dr. Miyaoka and colleagues summarize the current Medicine in Brussels, who pioneered the introduction of knowledgeontheimpactofseveralfactorsonsperminjection ICSI and revolutionized the treatment of male infertility in outcomesusingsurgicallyretrievedspermfrommenwithOA. the 90s. In our article, we summarized the data that have TheauthorsconcludethatSRinOAishighlysuccessfuland been generated on the reproductive potential of azoosper- that causes of obstruction and retrieval methods have little mic men undergoing assisted conception (15). We per- impact on SR success rates. Moreover, current evidence formed a systematic review of the literature focusing on suggests that similar pregnancy outcomes are achieved by studies that directly compared pregnancy outcomes after ICSIinOAusingeitherfreshorfrozen-thawedepididymalor sperm injections between couples whose male partner had testicular sperm (11). While a successful retrieval attempt is OAorNOA.Wealsoanalyzedapersonaldatabase(SCE)of obtained in virtually all cases of OA, Drs. Glina and Vieira 370 couples who underwent ICSI according to the above- highlighttheuncertaintyofspermacquisitionincasesofNOA, citedcategoriesandcomparedtheoutcomeswithagroupof thus making it desirable to determine the prognostic factors. 465 non-azoospermic infertile males. In our series of 1,092 Theauthorsreviewseveralclinicalandlaboratoryprognostic ICSI cycles performed in 835 male infertility patients, live markers,suchastheetiologyofNOA,paternalage,testicular birth rates were lowest in the NOA group. Miscarriage, volume, serum levels of pituitary gonadotropins, genetic ectopic pregnancy, and multiple pregnancy rates did not testingresults,methodofcollection,testicularhistopathology differ between clinical pregnancies achieved using ejacu- results and the impact of the laboratory tissue processing latedornon-ejaculatedspermfrommenwithOAorNOA. method; the authors concluded that the only unfavorable Inourseriesof427babiesbornwithICSIusingspermfrom indicatorforSRisthepresenceofmicrodeletionsintheAZFa non-azoospermic infertile fathers and azoospermic fathers and/or AZFb regions of the Y chromosome long arm (12). withOAandNOA,theshort-termneonataloutcomeswere AnotherkeymessagefromthisreviewisthatmenwithNOA similar among groups, despite a tendency towards higher are no longer considered sterile, even with elevated follicle- preterm birth in both azoospermia categories and lower stimulatinghormonelevelsandsmalltestes,becausemodern gestationalagefortwinsinOA.Theoverallperinataldeath retrievaltechniquescanbeusedtocollecttesticularspermand and malformation rates were 2.8% and 1.6%, respectively, produceahealthybiologicaloffspringviaassistedconception. and our results did not differ between deliveries that The laboratory management of surgically retrieved resultedfromICSIusingejaculatedornon-ejaculatedsperm gametes requires special attention because spermatozoa from men with OA or NOA. In our review, we critically collected from azoospermic men are often compromised in compare our results with other publications. We note that qualityandmorefragile.Drs.PopalandNagyfromAtlanta most published studies, including our data, suffer from provide strategies for handling such gametes inside the methodological shortcomings. For instance, these studies laboratoryanddiscusspotentialdangers(13).Adherenceto were not designed to detect differences in live birth rates stateoftheartlaboratorytechniquesandqualitycontrolare and not powered to detect differences in less frequent recommendedtoavoidjeopardizingthefertilizingpotential outcomes, such as malformations and other complications. of the sperm and chances of achieving a live birth. Several Moreover,nofollow-upstudyhasyetcomparedthelong-term 3 Editorialforthespecialissueonazoospermia CLINICS2013;68(S1):1-4 EstevesSandAgarwalA physical, neurological and developmental outcomes of chil- andtheproductionofhumangametesinthelaboratoryisa drenbornwithICSIusingspermfromazoospermicmenwith highly complex process that has yet to be translated to OAandNOA.Weconcludethatfornow,thelimitedevidence reproductive medicine. onpregnancyandpostnataloutcomesofICSIusingsurgically ThisSpecialIssueofClinicsaimstobealandmarktreatise derivedspermfromazoospermicmenisreassuring;however, on azoospermia. Scientists and clinicians from seven a call for continuous monitoring is of utmost importance to countries on three continents have contributed generously support the recommendation of sperm retrieval and ICSI in to the current scientific knowledge involving human azoospermia-relatedmaleinfertility.Followingthistheme,an azoospermia and its role in male reproductive health. We authoritativereviewbyDr.Veerleandco-authorsprovidesan recommend its contents not only to students and research- analysis of the results of immature germ cells used for ICSI ers in the biological, veterinary and medical sciences but (16). This strategy has been proposed in cases of NOA in also to clinicians involved in the management of infertile which no spermatozoa can be retrieved. After the initial couples and urologists, andrologists, gynecologists, embry- disappointing results, the in vitro culture of immature germ ologistsandreproductivespecialistsinterestedinfollowing cellstomorematurestageshasbeenproposedasanapproach the exponential growth in the knowledge of azoospermia. toimprovethispooroutcome.Morethanadecadehaspassed Duetothemultidisciplinarynatureofthiscategoryofmale since the introduction of ICSI with elongating and round infertility, unsolved problems present themselves, and the spermatids; there is still a lot of uncertainty regarding the opportunities for advancement continue to expand. We safetyofthistreatmentoption.Theauthorsoutlinetheclinical hopethatreaderswillappreciatethisSpecialIssueofClinics andscientificevidenceforICSIusingimmaturegermcellsand andshare our excitement inthestudy ofazoospermia. in vitro matured germ cells and describe the physiopatholo- gical mechanisms involved in fertilization. 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