Y染色体微缺失检测指导
EAA EMQN Y染色体微缺失诊断指南(2013版)
Correspondence:Csilla Krausz,Andrology Unit,Department of Experimental and Clinical Biomedical Sciences,Viale Pieraccini 6,Florence 50139,Italy.E-mail:c.krausz@dfc.unifi.it;Frank T €uttelmann,Institute of Human Genetics,University of M €unster,Vesaliusweg 12-14,M €u nster 48149,Germany.E-mail:frank.tuettelmann@ukmuenster.deKeywords:AZF,azoospermia,genetics,gr/gr deletion,male infertility,oligozoospermia,quality control,spermatogenesis,Y chromosome microdeletion Received:3-Nov-2013Revised:17-Nov-2013Accepted:18-Nov-2013doi:10.1111/j.2047-2927.2013.00173.xEAA/EMQN best practice guidelines for molecular diagnosisof Y-chromosomal microdeletions:state-of-the-art 20131C.Krausz,2L.Hoefsloot,3,4M.Simoni and 5F.T €uttelmann 1Andrology Unit,Department of Experimental and Clinical Biomedical Sciences,University ofFlorence,Florence,Italy,2Department of Human Genetics,Radboud University Nijmegen Medical Centre,Nijmegen,The Netherlands,3Department of Biomedicine,Metabolism and Neural Sciences,University of Modena and Reggio Emilia,4Azienda USL di Modena,NOCSAE,Modena,Italy,and 5Institute of Human Genetics,University of M €unster,M €u nster,Germany SUMMARYThe molecular diagnosis of Y-chromosomal microdeletions is a common routine genetic test which is part of the diagnosticworkup of azoospermic and severe oligozoospermic men.Since 1999,the European Academy of Andrology (EAA)and the European Molecular Genetics Quality Network (EMQN)have been actively involved in supporting the improvement of the quality of the diag-nostic assays by publication of the laboratory guidelines for molecular diagnosis of Y-chromosomal microdeletions and by offeringexternal quality assessment trials.The present revision of the 2004laboratory guidelines summarizes all the clinical novelties relatedto the Y chromosome (classic,partial and gene-specific deletions,genotype –phenotype correlations,methodological issues)and pro-vides an update on the results of the quality control programme.These aspects also reflect the consensus of a large group of special-ists present at a round table session during the recent Florence-Utah-Symposium on ‘Genetics of male infertility’(Florence,19–21September,2013).During the last 10years the gr/gr deletion has been demonstrated as a significant risk factor for impaired spermproduction.However,the screening for this deletion type in the routine diagnostic setting is still a debated issue among experts.Theoriginal basic protocol based on two multiplex polymerase chain reactions remains fully valid and appropriate for accurate diagnosisof complete AZF deletions and it requires only a minor modification in populations with a specific Y chromosome background.However,in light of novel data on genotype –phenotype correlations,the extension analysis for the AZFa and AZFb deletions is nowroutinely recommended.Novel methods and kits with excessively high number of markers do not improve the sensitivity of the test,may even complicate the interpretation of the results and are not recommended.Annual participation in an external quality control programme is strongly encouraged.The 12-year experience with the EMQN/EAA scheme has shown a steep decline in diagnostic(genotyping)error rate and a simultaneous improvement on reporting practice.INTRODUCTIONAfter the Klinefelter syndrome,Y-chromosomal microdele-tions are the second most frequent genetic cause of male infertil-ity.In the last decade,many investigators have described theoccurrence of microdeletions in infertile patients around the world and the molecular diagnosis of deletions has become animportant test in the diagnostic workup of male infertility (Vogtet al.,1996;Krausz &Degl’Innocenti,2006;Simoni et al.,2008).Microdeletions occur in about one in 4000men in the general population but its frequency is significantly increased among infertile men.Azoospermic men have a higher incidence ofmicrodeletions than oligozoospermic men and consequentlydeletion frequency found in different laboratories may vary from2to 10%(or even higher,Fig.1)reflecting the composition of the study population (Krausz et al.,2001;Simoni et al.,2008;LoGiacco et al.,2013).Typically,routine laboratories receiving referrals from outside institutions,without controlled patient selection,have a much lower incidence,<2%.The published data and the quality control programme experi-ence showed that diagnostic protocols can be quite different and©2013American Society of Andrology and European Academy of Andrology Andrology ,2014,2,5–195This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License,which permits use anddistribution in any medium,provided the original work is properly cited,the use is non-commercial and no modifications or adaptations are made.ISSN:2047-2919ANDROLOGY诊断检查无精子症严重少精子症的男性欧洲男科协会欧洲分子遗传质量网络诊断分析实验室指南发表分子诊断外部质量评估试验概述临床的特点方法论问题质量管理程序方面反映专家会议研讨会男性不育遗传学证明重要的危险因素被损害的精子产生筛选日常诊断环境辩论问题专家，内行原始的基础方案多重PCR 适合的准确诊断完全的修改修饰根据新的数据基因型与表现型的相互关系扩展分析常规推荐的过分的，非常的敏感度弄复杂解释推荐每年参与方案陡峭的下降诊断错误率基因分型检查无精子症发病率少精子症合乎逻辑的实验室变化反映出著作代表性地诊断治疗方案that inaccurate or wrong diagnoses occur as well,suggesting thenecessity of both standardization and quality control (Simoni,2001).Therefore,the European Academy of Andrology (EAA)and the European Molecular Genetics Quality Network (EMQN)jointly supported the publication of two ‘Laboratory guidelinesfor molecular diagnosis of Y-chromosomal microdeletions’(Simoni et al.,1999,2004)and started offering external quality assessment (EQA).During the last 9years,novel data concerning gene-specific deletions,partial AZFc deletions/duplications and genotype –phenotype correlations have been accumulated.All these issues together with some methodological aspects which urged to be clarified,and an update on the EAA/EMQN AZF quality control scheme’s activity is summarized in this study.STRUCTURE OF THE MALE-SPECIFIC REGION OF THE Y CHROMOSOME (MSY)The complete physical map and sequence of MSY have been available since 2003(Skaletsky et al.,2003).This information was obtained by sequencing and mapping 220BAC clones containing portions of the MSY from one man.The use of only one individual was necessary because,owing to the presence of repetitive sequences with only minute differences characterizing the individual copies of each sequence (sequence family vari-ants,SFV),interindividual allelic variation or polymorphisms would have prevented the accurate mapping of SFV necessary to allocate the BAC clones.Three classes of sequences were found in MSY:X-transposed (with 99%identity to the X chromosome),X-degenerate (single-copy genes or pseudogene homologues of X-linked genes)and ampliconic.Ampliconic sequences are char-acterized by sequence pairs showing nearly complete (>99.9%)identity,organized in massive palindromes.According to current knowledge,the reference MSY contains 156transcription units including 78protein-coding genes encoding 27proteins.Ampli-conic sequences comprise 60coding genes and 74non-coding transcription units mostly grouped in families and expressed mainly or only in the testis.Ampliconic sequences recombine through gene conversion,that is,non-reciprocal transfer of sequence information occurring between duplicated sequences within the chromosome,a process which maintains the >99.9%identity between repeated sequences organized in pairs in inverted orientation within palindromes.Besides maintaining the gene content,this peculiar sequence organization provides the structural basis for deletions and rear-rangements.It is widely accepted that complete AZF deletions arise invariably through Non-Allelic Homologous Recombina-tion (NAHR)which takes place between highly homologous repeated sequences with the same orientation leading to loss of the genetic material between them.Considering the architecture of the MSY,several different deletions are hypothetically possi-ble (Kuroda-Kawaguchi et al.,2001;Yen,2001;Repping et al.,2003)and those which are clinically relevant for male infertility,based on current knowledge,are briefly described below.MECHANISM AND TYPE OF DELETIONSThree discrete AZFa ,AZFb and AZFc regions were originally characterized by careful mapping of the MSY of a large number of men with microdeletions when the sequence of the Y chromo-some was not completely known (Vogt et al.,1996).Subse-quently,thanks to the fine molecular characterization of the deletions,a new model of deletions,in which the AZFb and AZFcregions are overlapping,have been proposed (Fig.2).In addi-tion,the AZFb and AZFbc deletions have been suggested to bethe consequence of at least three different deletions patterns(Repping et al.,2002)(Fig.2).While the new nomenclature ismore appropriate in biological terms,from the practical,clinical point of view either nomenclature can be adopted for the com-plete AZFb (P5/proximal P1)and AZFc (b2/b4)deletions.On theother hand,the distinction between the two AZFbc subtypes (P5/distal P1and P4/distal P1)does have clinical relevance (see below).We additionally provide information on the genomic localization of the sY-loci used for the AZFanalysesFigure 1Worldwide frequencies of AZF deletions in infertile men (reprinted from Simoni et al.,2008with publisher’s permission).Percentages are coded in colours according to the legend.Note:Sweden,Germany and Austria show the lowest incidence.However,the composition of the study populations dif-fered in terms of the proportion of azoospermic vs.oligozoospermic patients which may also contribute to the observed differences.6Andrology ,2014,2,5–19©2013American Society of Andrology and European Academy of AndrologyC.Krausz et al.ANDROLOGY不准确标准化Y染色体微缺失实验室诊断指南外部质量评估完整的物理图谱Y染色体上男性特定区域结构机制分型重叠的后果命名法适当的生物学术语实际的临床采用区分亚型临床关联性(Appendix C),which should be used to describe deletions fol-lowing the HGVS nomenclature as part of a standard practice.The description of the AZFc (b2/b4)deletion is given in ‘report examples’.The AZFa region is about 1100kb long and contains the sin-gle-copy genes USP9Y (former DFFRY )and DDX3Y (formerDBY).Recent data obtained simultaneously by different groupsidentify the origin of complete AZFa deletions in the homolo-gous recombination between identical sequence blocks withinthe retroviral sequences in the same orientation HERVyq1and HERVyq2(Blanco et al.,2000;Kamp et al.,2000;Sun et al.,2000).Within these retroviruses,recombination can occur in either one of two identical sequence blocks (ID1and ID2),giving rise to two major pattern of deletions slightly different in their precise breakpoints (Kamp et al.,2000;Sun et al.,2000;Kamp et al.,2001).In any case,the complete deletion of the AZFa region removes about 792kb including both USP9Y and DDX3Y genes,the only two genes in the AZFa region.The type and mechanism of deletions of the AZFb and AZFc region have been clarified by Kuroda-Kawaguchi et al.(2001).Both regions together comprise 24genes,most of which are present in multiple copies for a total of 46copies.The complete deletion of AZFb removes 6.2Mb (including 32copies of genes and transcription units)and results from homologous recombi-nation between the palindromes P5/proximal P1(Repping et al.,2002).The AZFc region includes 12genes and transcription units,each present in a variable number of copies making a total of 32copies (Repping et al.,2003).The classical complete dele-tion of AZFc,the most frequent pattern among men with dele-tions of the Y chromosome,removes 3.5Mb,originates from the homologous recombination between amplicons b2and b4in palindromes P3and P1,respectively,and removes 21copies of genes and transcription units (Kuroda-Kawaguchi et al.,2001).Deletions of both AZFb and AZFc together occur by two major mechanisms involving homologous recombination between P5/distal P1(7.7Mb and 42copies removed)or between P4/distal P1(7.0Mb,38copies removed)(Repping et al.,2002).Therefore,according to the present knowledge,the followingrecurrent microdeletions of the Y chromosome are clinically rel-evant and are found in men with severe oligo-or azoospermia (Fig.2):•AZFa,•AZFb (P5/proximal P1),•AZFbc (P5/distal P1or P4/distal P1),•AZFc (b2/b4).The most frequent deletion type is the AZFc region deletion (~80%)followed by AZFa (0.5–4%),AZFb (1–5%)and AZFbc (1–3%)deletion.Deletions which are detected as AZFabc are most likely related to abnormal karyotype such as 46,XX male or iso(Y)(Lange et al.,2009).Gr/gr deletion The AZFc region is particularly susceptible to NAHR eventswhich may cause the formation of both partial deletions andduplications leading to gene dosage variations (Kuroda-Kawagu-chi et al.,2001;Yen,2001;Krausz et al.,2011).Although a num-ber of different partial AZFc deletions have been described,only one of them is of potential clinical interest.This is the ‘gr/gr’deletion,named after the fluorescent probes (‘green’and ‘red’)used when first described (Repping et al.,2003).Although it removes half of the AZFc gene content (genes with exclusive or predominant expression in the germ cells),its clinical signifi-cance is still a matter of debate,because carriers may exhibit highly variable spermatogenic phenotypes ranging from azoo-to normozoospermia.Clearly the effect of the deletion is largely dependent on the ethnic and geographic origin of the study pop-ulation.In fact,the frequency and phenotypic effect may vary among different ethnic groups,on the basis of the Y chromo-some background;for example,in specific Y haplogroups,such as D2b,Q3and Q1,common in Japan and certain areas of China,the deletion is fixed and apparently does not have negative effects on spermatogenesis (Sin et al.,2010;Yang et al.,2010).Controversies are also related to selection biases (lack of ethnic/geographic matching of cases and controls;inappropriate©2013American Society of Andrology and European Academy of AndrologyAndrology ,2014,2,5–197GUIDELINES FOR Y-MICRODELETION TESTING 2013ANDROLOGY近端远端临床上严重的寡核苷酸无精子症特别敏感的形成局部缺失重复剂量变化附录同时发生同源重组反转录病毒序列重组发生包含selection of infertile and control men)and methodological issues (lack of confirmation of gene loss).Many efforts have been done to clarify the molecular basis for the highly variable phenotypic presentation of this deletion type.It has been previously described that the loss of DAZ1/DAZ2and CDY1is prevalent (or even specific)in carriers with impaired sperm production (Fer-nandes et al.,2002;Ferlin et al.,2005;Giachini et al.,2005)while it was hypothesized that the restoration of normal AZFc gene dosage in case of gr/gr deletion followed by b2/b4duplication may explain the lack of effect on sperm count (Repping et al.,2003).In this regard,a large multicentre study,based on a com-bined method (gene dosage,definition of the lost DAZ and CDY1genes,Y hgr definition),was performed on Caucasians (Krausz et al.,2009).Notwithstanding the detailed characterization of subtypes of gr/gr deletions based on the type of missing gene copies and the detection of secondary rearrangements (deletion followed by b2/b4duplication)together with the definition of Y haplogroups,it was impossible to define a specific pattern which would be associated with either a ‘neutral’or a ‘pathogenic’effect.On the contrary,studies dealing with Asian populations seem to support the hypothesis about a deletion subtype-depen-dent phenotypic effect and about the importance of the Y back-ground on which the deletion arises (Yang et al.,2010;Choi et al.,2012).In addition to classic case/control studies aiming to define whether the gr/gr deletion confers a risk for spermato-genic disturbances,the analysis of consecutive patients through cross-sectional cohort analysis indicates that the gr/gr deletion has an effect even within the normal range of sperm count.It was observed,indeed,that normozoospermic carriers have a sig-nificantly lower sperm count,compared to men with intact Y chromosome (Visser et al.,2009).In addition,Yang et al.(2006)reported that,in the Chinese population,the deletion frequency drastically decreases in subgroups with sperm concentrations >509106/mL.The screening for gr/gr deletion is based on a PCR plus/minus method of two markers (sY1291and sY1191)(Repping et al.,2003)and the diagnosis is based on the absence of marker sY1291and presence of sY1191.It is worth noticing that a 5%false deletion rate has been detected in the multicenter study (Krausz et al.,2009),underlining the importance of the optimi-zation of the PCR conditions and of additional confirmatory steps such as simplex PCR and eventually gene dosage analysis (Giachini et al.,2005;Choi et al.,2012).The definition of the Y haplogroup is indicated in Asian patients to exclude constitutive deletions which are unlikely to affect spermatogenesis (see above).Clinical implicationsAs stated above,the heterogeneity of the study populations available in the literature complicates a reliable meta-analysis.However,four meta-analyses have been attempted on this topic all achieving significant odds ratios reporting on average 2-to 2.5-fold increased risks of reduced sperm output/infertility (T €uttelmann et al.,2007;Visser et al.,2009;Navarro-Costa et al.,2010;Stouffs et al.,2011).Therefore,the gr/gr deletion repre-sents a unique example in andrology of a confirmed significant genetic risk factor for impaired sperm production.For instance,in the Italian population,gr/gr deletions confers a 7.9-fold increased risk for spermatogenic impairment (OR =7.9,95%CI 1.8–33.8)(Ferlin et al.,2005;Giachini et al.,2008).A gr/gr deletion (i.e.a genetic risk factor for impaired sperm production)will be obligatorily transmitted to the male off-spring.The partial deletion may expand to a complete AZFc deletion (i.e.a clear-cut causative factor for spermatogenic impairment)in the next generations (Zhang et al.,2007),but data are currently sparse to draw final conclusions on this spe-cific risk.Gr/gr deletions have also been proposed as genetic risk factor for testis cancer (Nathanson et al.,2005;Linger et al.,2007)but this association still awaits further confirmation on large independent study populations.The low cost of the test may justify its routine testing in those populations for which robust and consistent data with risk esti-mate are available (at present Italian,Spanish,Dutch and Chi-nese).Currently,however,no general agreement to adviseroutine testing has been reached (T €uttelmann et al.,2007;Krausz et al.,2011;Stouffs et al.,2011).Isolated AZF gene-specific deletionsAlthough some authors found an extraordinary high frequency of single AZF gene deletions (Ferlin et al.,1999;Foresta et al.,2000),these data are in stark contrast with the general experi-ence accumulated in >2000patients tested elsewhere (Silber et al.,1998;Sun et al.,1999;Krausz et al.,1999a,b;Krausz et al.,2001;Simoni et al.,2008).Gene-specific deletions are extremely rare,and all the five confirmed deletions (with the definition of the breakpoints)removed totally or partially the USP9Y gene belonging to the AZFa region (Tyler-Smith &Krausz,2009).None of the deletions was because of NAHR and thus are likely to be unique,supporting the extreme rarity of the occurrence of these events.The associated semen/testis phenotype is largely variable among USP9Y deletion carriers (from azoospermia caused by hypospermatogenesis to normozoospermia)indicating that this gene rather acts as a fine tuner than an essential factor for sper-matogenesis.Based on the absence of other than USP9Y gene deletions in the literature,screening for isolated gene-specific deletions is not advised in the routine diagnostic setting.Given that some of the commercially available kits contain gene-spe-cific markers,much care has to be taken both of the validation of suspected single-gene deletions as well as of the interpreta-tion of the results.Genotype/phenotype correlation of complete AZF deletions AZF deletions are specific for spermatogenic failure as no deletions have been reported in a large number of normozoo-spermic men (Krausz et al.,2003;Simoni et al.,2008).Although ‘fertility’can be compatible with these deletions,it simply reflects the fact that natural fertilization may occur even with low sperm counts depending on the female partner’s fertility status.For this reason,it is more appropriate to consider Y deletions as a cause of oligo/azoospermia rather than a cause of ‘infertility’.Deletions of the entire AZFa region invariably result in ser-toli cell only syndrome (SCOS)and azoospermia (Vogt et al.,1996;Krausz et al.,2000;Kamp et al.,2001;Hopps et al.,2003;Kleiman et al.,2012).The diagnosis of a complete deletion of the AZFa region implies the virtual impossibility to retrieve testicular spermatozoa for intracytoplasmic sperm injection (ICSI).Complete deletions of AZFb and AZFbc (P5/proximal P1,P5/distal P1,P4/distal P1)are characterized by a histological picture8Andrology ,2014,2,5–19©2013American Society of Andrology and European Academy of AndrologyC.Krausz et al.ANDROLOGY单独的临床意义如上所述异质性of SCOS or spermatogenetic arrest resulting in azoospermia.Several reports have shown that similar to the complete dele-tions of the AZFa region,no spermatozoa are found upon attempts of testicular sperm extraction (TESE)in these patients (Krausz et al.,2000;Hopps et al.,2003;Kleiman et al.,2011).However,in three cases,spermatid arrest and even crypto/oligo-zoospermia has been reported in association with complete AZFb or AZbc deletions (Longepied et al.,2010;Soares et al.,2012).The biological explanation of the unusual phenotypes remains unclear,both Y background effect and differences in the exact extent of the deletions may account for it.In fact,a smaller deletion,that is,a proximal breakpoint at P4may be associated with the retention of AZFb gene copies such as XKRY ,CDY2and HSFY .It has been therefore proposed that the associated pheno-type is more severe in case of complete removal of the AZFb region.With very few exceptions reported in the literature,the diagnosis of complete deletions of AZFb or AZFbc (P5/proximal P1,P5/distal P1,P4/distal P1)implies that the chance for testicu-lar sperm retrieval is virtually zero even with micro-TESE (Bran-dell et al.,1998).Deletions of the AZFc region (b2/b4)are associated with a variable clinical and histological phenotype (Reijo et al.,1996;Luetjens et al.,2002;Oates et al.,2002).In general,AZFc dele-tions are compatible with residual spermatogenesis and thus can be found also in men with severe oligozoospermia and,in rare cases,may even be transmitted naturally to the male off-spring (K €uhnert et al.,2004and references therein).In men with azoospermia and AZFc deletion there is approximately 50%chance of retrieving spermatozoa from TESE and children can be conceived by ICSI (Kent-First et al.,1996;Mulhall et al.,1997;Kamischke et al.,1999;Jiang et al.,1999;Kleiman et al.,1999;Page et al.,1999;Cram et al.,2000;van Golde et al.,2001;Oates et al.,2002;Peterlin et al.,2002;Ferlin et al.,2007;Simoni et al.,2008;Lo Giacco et al.,2013).The TESE success rate largely depends on the technique used and can be as low as 9%(Lo Gi-acco et al.,2013)and as high as 70–80%following micro-TESE (Hopps et al.,2003).According to one report,the presence of 45,X cell lines in blood may be a negative predictive factor for spermatogenesis (Jaruzelska et al.,2001).INDICATIONS FOR MOLECULAR SCREENING OF THE Y CHROMOSOMEDiagnosis of a microdeletion of the Y chromosome permits the cause of the patient’s azoospermia/oligozoospermia to be established and to formulate a prognosis.In which patients should molecular screening of the Y chromosome be per-formed?The world literature,now based on several thousands of patients screened,indicates that,as a rule,clinically relevant deletions are found in patients with azoospermia or severe oli-gozoospermia with sperm concentrations <29106/mL.Very rarely,deletions can be found in infertile patients with sperm concentration between 2and 59106/mL (Maurer &Simoni,2000;Lo Giacco et al.,2013).We provide a flow chart with these indications and including the recommended analytic steps in Fig.3.The usual clinical parameters such as hormone levels,testicular volume,varicocoele,maldescended testis,infections,etc.do not have any predictive value (Maurer et al.,2001;Oates et al.,2002;Tomasi et al.,2003;Simoni et al.,2008).In general,molecular analysis of the Y chromosome is not indicated in patients with chromosomal abnormalities (except 46,XY/45,Xkaryotype),obstructive azoospermia (unless FSH is above the normal limit)or hypogonadotropic hypogonadism.However,in the literature there are a number of examples of deletion carri-ers among non-idiopathic infertile men,for example,with a testicular tumour or after chemo-/radiotherapy,which would be considered to explain the spermatogenic failure.Therefore,the presence of any diagnosis accompanied by azoo-or severe oligozoospermia should be an indication for AZF testing.For instance,in men belonging to the above semen categories,AZF screening is important before varicocoelectomy because dele-tion carriers will most likely not benefit from the surgical procedure.After a high incidence of AZF deletions in Klinefelter patients had been reported in two small studies (Mitra et al.,2006;Had-jkacem-Loukil et al.,2009),the question arose whether deletion screening should be routinely be performed in these men.However,the described deletions were mainly diagnosed by only isolated markers of the AZFa and/or the AZFb region,not con-firmed by additional analyses,and should probably be regarded as methodological artefacts.In contrast,three much larger stud-ies did not find any AZF deletions in Klinefelter patients (Choe et al.,2007;Simoni et al.,2008;Rajpert-De Meyts et al.,2011).Patients with azoospermia who may be candidate for TESE/ICSI should be offered deletion screening because TESE should not be recommended in cases of complete deletion of the AZFa region.Micro-TESE in azoospermic carriers of deletions of the AZFb or AZFbc regions with proximal breakpoint in the P4palin-drome may be eventually attempted.However,the patient should be fully informed about the very low/virtually zero chance to retrieve spermatozoa.A standard biopsy (without microsurgical equipment)should never be attempted in these cases.Therefore,the diagnosis of a deletion has prognostic value and can influence therapeutic options.GENETIC COUNSELLINGGenetic counselling is mandatory to provide information about the risk of conceiving a son with impaired spermatogene-sis.In case of partial AZFa or AZFb and AZFc deletion,the coun-selling (with AZF testing)is relevant also for other male members of the family as transmission of these type of deletions has been reported in the literature (Krausz et al.,2006;Luddi et al.,2009;Plotton et al.,2010).Complete AZFa,AZFb,AZFbc or AZFabc deletion are generally incompatible with sperm production thus family screening is not indicated.Several studies have been published about ICSI performed in couples with male partners carrying AZFc deletions (Kent-First et al.,1996;Mulhall et al.,1997;Jiang et al.,1999;Kami-schke et al.,1999;Kleiman et al.,1999;Page et al.,1999;Cram et al.,2000;van Golde et al.,2001;Oates et al.,2002;Peterlin et al.,2002;Stouffs et al.,2005;Mau Kai et al.,2008;Simoni et al.,2008;Mateu et al.,2010;Lo Giacco et al.,2013).Although a lower fertilization rate and embryo quality (van Golde et al.,2001),a significantly impaired blastocyst rate (Mateu et al.,2010)and lower overall success of ICSI (Simoni et al.,2008)have been reported,the majority of studies report no signifi-cant differences in fertilization and pregnancy rates between men with or without Y deletion.While the deletion of the father will be obligatory transmitted to the son,who will have impaired sperm production,the exact testicular phenotype cannot be predicted because of the different genetic©2013American Society of Andrology and European Academy of AndrologyAndrology ,2014,2,5–199GUIDELINES FOR Y-MICRODELETION TESTING 2013ANDROLOGY遗传咨询适应证微缺失筛查。
Y染色体微缺失
好的解释发病原因，提供遗传咨询和指导临床诊疗。
• 1996年将 AZF区划分为 3个区:AZFa、AZFb、 AZFc。1999 年研究发现在 AZFb 和 AZFc 间存在AZFd。研究发现 ,在 Y染色体 AZFa、 AZFb、 AZFc3个区至少发现 15 个与精子发生相关的基因，AZFd区尚未发现相关基因。
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四 Ｙ 染色体微缺失的检测方法
• 选择位于 Y染色体短臂 ( Y p ) 的睾丸决定基因( S RY ) 和锌 指蛋白基因( Z F X ／ Z F Y ) 为对照。Z F X ／ Z F Y既存在于 x染色体短臂， 也存在于 Y p ， 其与 S R Y一起构成2个 目 前推荐使用 的参照基因。每个 A Z F区域至少设置2个 S T S 位点 才能正确有效.
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二 Y染色体微缺失相关基因
• AZFa缺失的病人 , 主要表现为唯支持细胞综 合征伴睾丸体积缩小 ,无精子生成
• AZFb缺失的病人主要表现为生精过程阻滞 在精母细胞阶段 ,无精子生成
• AZFc区缺失病人表现多样化 ,会出现无精子 症和少精子症的不同临床表现
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二 Y染色体微缺失相关基因
• AZFa缺失患者 33%为严重少精子,67%表现为 无精子
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四 Ｙ 染色体微缺失的检测方法
• 由于不 同实验室采用 P C R方法不 同,导致 实验结果各异。为提高诊断质量， 欧洲男 科协 会( E A A ) 和欧洲分子遗传实验质控网 ( E MQ N) 在 1 9 9 9 、 2 0 0 4年先后颁布了 第 1版和第 2版 Y染色体 微缺失分子诊断指 南， 规范了检测微缺失时的Y 染色体序列 标签点( s e q u e n c e t a r g e t e d s i t e s ， S T S ) 与 所采用的内外对照方法。
Y 染色体微缺失快速检测系统 说明书
Y染色体微缺失快速检测系统说明书 ２５人份／盒z概述本试剂盒用于快速检测人（男性）Y染色体AZF座位（Azoospermia Factor）的缺失。
多项研究发现，AZF基因家族共有AZFa、AZFb、AZFc三个座位（在AZFb与AZFc之间还存在AZFd座位，中等程度的少精和精子数目正常却形态异常多会与该区域的缺失有关），其中任何一个座位的微缺失都可导致生精障碍。
本试剂盒共设计15对引物和一对内控（内部质量控制）引物，利用PCR（Polymerase Chain Reaction）技术可从Y染色体上特异性扩增出15条非多态性短片段的序列标签位点（Sequence Tagged Sites，STS）和一条内控基因片段。
这些引物对分别组合成4套多重PCR系统，在相同条件下，进行4管PCR扩增反应，通过电泳结果判定15个序列标签位点（STS）的存在与否。
主要特点：1.可对15个STS序列标签位点进行检测，这15个位点覆盖了所有的AZF区域，其中AZFa有3个位点，AZFd有2个，AZFb有6个，AZFc 有4个位点；2.高特异引物确保结果的可靠性，并具有内控（内部质量控制）基因片断的检测；3.独特的防污染设计，有效避免假阳性的产生；4.符合并超过欧洲生殖学会推荐的检测标准。
应用：胞质内单精子注射（ICSI）技术辅助生育治疗前的检测、少精症与无精症男性患者遗传学检测、精子库的入库前筛查等。
z试剂盒组成组成成分数量保存条件PCR反应液Ⅰ（紫色） 25管(每管14μL ) -20℃PCR反应液Ⅱ（无色） 25管(每管14μL) -20℃PCR反应液Ⅲ（蓝色） 25管(每管14μL) -20℃PCR反应液Ⅳ（黄色） 25管(每管14μL) -20℃纯水z有效期六个月z需自备的仪器及试剂PCR仪（推荐使用PTC-100或PTC-200, M J Research Co.或其它国际知名品牌PCR仪）DNA电泳系统z操作步骤1.基因组DNA的获取使用本公司全血DNA快速提取试剂盒从抗凝全血中抽提人（男性）基因组DNA，按操作说明进行。
PCR—PAGE用于Y染色体微缺失的检测
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Y染色体微缺失检测
Y染色体微缺失检测试剂盒背景介绍根据国际卫生组织调查数据，约15%育龄夫妇存在生育障碍，其中男性因素引起的生育障碍约占一半。
在已知的导致男性不育的遗传学因素中，发病率最高的两种是Y染色体的微缺失和克氏综合症。
Y染色体具有大量重复基因序列及回文结构，这些结构在维持Y染色体进化稳定性的同时也使回文结构内部基因易于丢失，进而导致不育。
缺失率最高的三个影响精子发生的区域被命名为AZFa，AZFb和AZFc，它们之中任何一个出现缺失都有可能导致育性下降或不育。
Y染色体微缺失在无精症或少精症患者中发病率在10-15%，已成为男性不育患者的常规检查项目。
图1，Y染色体AZF区结构示意图（AZFa区两端有两段原病毒序列、AZFb/c区内部有5个序列高度一致的大的回文结构，这些序列和结构导致相应染色体片段易发生同源重组，造成缺失或复制。
）欧洲男科学协会和欧洲分子遗传实验质控协作网（EAA/EMQN）2004年发表“Y染色体微缺失分子诊断指导意见”建议通过对各AZF区的共6个STS位点和两个对照位点检测Y染色体微缺失。
产品简介本剂盒通过“多重定量荧光PCR技术”（Multiplex Quantitative Fluorescent PCR），以一个高度复合的扩增体系中扩增至少15个Y染色体微缺失检测相关的位点，并根据各位点有无扩增产物及扩增产物剂量判断缺失类型。
对于Y染色体微缺失，由于各AZF区片段相对较小，常规核型分析等方法难以发现其缺失。
本项目采用的检测方法是：在各AZF区上分别选择具有序列特异性的多个位点，通过多重定量荧光PCR方法检测各位点。
根据有无扩增产物判定样本染色体是否包含所检测的序列特异性位点，进而推断样本染色体是否在位点所在AZF区域发生缺失；通过部分位点扩增产物相对剂量确定相关位点拷贝数比例，根据拷贝数比例推断相关位点对应区域是否发生缺失或复制，并对于AZFb和/或AZFc区部分缺失或复制，实现了首次检测。
快速检测Y染色体微缺失方法的建立
第 6 卷 第 4 期2020 年 8 月生物化工Biological Chemical EngineeringVol.6 No.4Aug. 2020快速检测Y染色体微缺失方法的建立杨神州（复旦大学 生命科学学院，上海 200438）摘要：为了快速检测Y染色体微缺失，基于重组聚合酶扩增技术，选取6个特异性标签位点（AZFa：sY84，sY86；AZFb：sY127，sY134；AZFc：sY254，sY255）和sY14内参为检测对象，分别用正常男性和正常女性样本验证。
结果表明该方法能在 1 h内检测出结果。
关键词：重组聚合酶扩增；Y染色体微缺失；快速检测中图分类号：Q819 文献标志码：ARapid Detection of Y Chromosome MicrodeletionsYang shen-zhou(School of Life Sciences Fudan University, Shanghai 200438)Abstract: In order to detect Y chromosome microdeletions rapidly, 6 Specific tagging sites(AZFa:sY84, sY86; AZFb: sY127, sY134; AZFc: sY254, sy255) were selected based on recombinant PCR and sY14 were used as the test objects, and were tested with normal male and normal female samples respectively. The results show that the method can detect the results within one hour.Key words: Recombinase Polymerase Amplification Y chromosome microdeletions detection据世界卫生组织报道，全球约1/10的育龄期夫妇不能正常生育后代，主要原因之一是男性因素引起的不育[1]。
11 Y染色体微缺失检测_8.15
Y染色体微缺失检测张磊博士深圳亚能医学检验所目录•背景知识•临床意义及适应症•亚能医学检验服务介绍•临床案例分享•其它服务简介背景知识1★据WHO数据统计，全世界有10~15%的夫妇患有不孕不育症;★其中男性因素占50%;★遗传因素占男性不育的30%;★在精子发生障碍引起的男性不育患者中，Y染色体微缺失的发生率仅次于克氏综合征，是居于第二位的遗传因素。
图1: 原发性不孕全球发病率统计（WHO, 2010年数据）图2: 继发性不孕全球发病率统计（WHO, 2010年数据）EAA/EMQN发布2013版指南12背景知识1Y染色体微缺失Y染色体长臂存在精子发生相关的基因，被称为无精子因子（AZF）；缺失率最高的三个影响精子发生的区域被命名为AZFa，AZFb和AZFc，它们之中任何一个出现缺失都有可能导致不育症。
3据WHO统计，男性原发性无精子症与少精子症患者中约10%~15%存在Y染色体微缺失。
背景知识1在中国，约有11.5%的男性不育是由AZF 区缺失引起的。
资料来源：全球不育男性中AZF缺失所占比例（Simoni et al., 2008 发行）Y染色体微缺失4背景知识1Y染色体AZF区结构示意图资料来源：Y染色体结构及微缺失模型（Repping et al., 2002）Ratio ~80%0.5-4%1-5%1-3%背景知识1AZFa区缺失AZFc区缺失AZFb区缺失AZFa、AZFb和AZFc缺失患者主要表现为唯支持细胞综合征（SCO综合征）伴睾丸体积缩小,无精子生成，占整个AZF缺失0.5~4%患者主要表现为生精过程阻滞在精母细胞阶段,无精子生成，占整个AZF缺失1~5%患者表现多样化,会出现无精子症和少精子症的不同临床表现，临床上最常见，约80%患者表现为100%无精子症临床表现Y Y染色体AZF区微缺失资料来源：2013年EAA/EMQN 指南（C. Krausz, et al., 2013）1资料来源：2013年EAA/EMQN 指南（C. Krausz, et al., 2013）2013年EAA/EMQN 指南AZF 区基础分析指南建议进行AZF区筛查流程背景知识AZF区筛查流程1资料来源：2013年EAA/EMQN 指南（C. Krausz, et al., 2013）2013年EAA/EMQN 指南AZF 区扩展分析背景知识临床意义及适应症2Y染色体微缺失检测临床意义3减少病人痛苦，提高辅助生殖成功几率1确定无精、少精患者病因，避免不必要的药物及手术治疗2尽量避免将有缺陷基因传递给下一代4为未来的基因治疗提供理论依据资料来源：2013年EAA/EMQN指南（C. Krausz, et al., 2013）2临床意义及适应症Y染色体微缺失检测适应症常规适应症☆男性不育症IVF、ICSI治疗前☆非梗阻性无精子症患者☆严重少精症者（少于5×106/mL）☆精子库入库前质量筛选☆无精症患者术前☆原因不明的男性不育症用药前资料来源：2004年EAA/EMQN指南（M. SIMONI, et al., 2004）2临床意义及适应症Y染色体微缺失检测适应症推荐适应症☆少精子症患者（精子数目少于20×106/mL）☆精子密度正常，但原因不明的男性不育症患者☆男性不育伴隐睾和精索静脉曲张的患者☆妻子有不明原因习惯性流产的患者资料来源：2004年EAA/EMQN指南（M. SIMONI, et al., 2004）二、STS-多重定性PCR法（EAA 和EMQN 推荐使用）一、实时荧光PCR法（简便、快速）三、基因芯片法四、荧光原位杂交技术Y染色体微缺失检测方法亚能医学检验服务介绍3服务介绍—PCR-荧光探针法3采用多重PCR结合多色Taqman荧光探针技术，选择与男性不育高度相关的Y染色体AZF区域6个序列标签位点（EAA/EMQN指南推荐）进行检测，以判断AZF区域是否存在微缺失。
对外周血Y染色体AZF微缺失基因检测-病理学论文-基础医学论文-医学论文
对外周血Y染色体AZF微缺失基因检测-病理学论文-基础医学论文-医学论文——文章均为WORD文档，下载后可直接编辑使用亦可打印——不育不孕症严重影响到一个家庭的和睦及幸福，随着科技及化工业的发展，不育不孕症患者越来越多，成为本世纪严重危害人类生殖健康的疾病之一。
据世界卫生组织（WHO）统计，全世界育龄夫妇约有15%存在生育问题（WHO1987年），这部分人群中有50%不育与男性因素有关，而由遗传缺陷因素所引起的生精障碍约占男性不育因素的30%。
男性不育患者中Y染色体长臂无精子症因子（azoos-permiafactor，AZF）区微缺失被认为是最重要的非梗阻性无精及少精症因素之一，现代分子生物学和细胞遗传学的研究亦证实了与精子生成相关的AZF基因存在于Y染色体长臂q11区，目前将Y染色体AZF区分为4个区位，分别为：AZFa区、AZFb区、AZFc区和AZFd区，其中任何一个区域座位点的微缺失都可导致生精障碍，造成少精或无精而导致男性不育，其微缺失引起的男性不育发生率仅次于Klinefelter综合征，并且在进行辅助生殖技术卵胞质内单精子注射（ICSI）过程中能将这种遗传缺陷传递给男性下一代，因此在进行ICSI治疗之前进行Y染色体微缺失检测具有重要的临床及理论意义。
本研究对来我院诊治的332例男性精子异常患者及100名已育男性进行外周血Y染色体AZF微缺失基因检测，现总结分析报告如下。
1 资料与方法1.1 临床资料病例组：选择2011年7月至2014年1月到我院生殖中心诊治的不育男性患者（年龄20～46岁），诊断标准：女方检查正常，结婚1年以上，未采取避孕措施且夫妻生活正常的情况下不能生育的非阻塞性男性患者，在排除了其他原因如感染等原因后对其进行精液常规分析，按照WHO精液分析标准分类：精子密度20106/mL者为少精症患者；患者每间隔7~14d留取精液检测1次，连续3次未发现精子，进一步离心沉淀也没有发现精子者为无精症患者。
中国人Y染色体微缺失分子诊断指南(草案)
中国人Y染色体微缺失分子诊断指南（草案）2005.4 上海前言在精子发生障碍引起的男性不育患者中，Y染色体微缺失的发生率仅次于Klinefelter’s syndrome(克氏综合征)，是居于第二位的遗传因素。
Y染色体微缺失已成为男性不育患者的常规检查项目。
欧洲男科学协会和欧洲分子遗传实验质控协作网为提高诊断质量，在1999年和2004年先后发布了第一版和第二版Y染色体微缺失分子诊断指南。
经过多年的临床实践证明该指南准确、灵敏和易于操作。
2005年4月在上海召开了中国人Y 染色体微缺失分子诊断的研讨会，成立了Y染色体微缺失分子诊断协作网。
会议在欧洲指南的基础上起草了符合目前我国男性不育诊疗现状，并反应最新生物技术发展的中国人Y染色体微缺失分子诊断指南。
本指南重点讨论Y染色体微缺失分子诊断具体实施时的标准化和规范化，推荐的相关方法和设计是根据欧洲指南和我国已有的实验研究基础上综合而成。
对机理研究和背景知识介绍部分在本指南中不再详细讨论。
男性不育症患者Y染色体微缺失分子筛查适应症常规检测的适应症：1、男性不育症患者选择单精子卵泡浆内注射（ICSI）或体外受精（IVF）生育子代前；2、非梗阻性无精子症患者；3、严重少精子症患者（精子数目少于5×106/ml）；4、无精子症患者进行睾丸活检术前；5、男性不育症患者（如精索静脉曲张）手术前；6、原因不明的男性不育症患者用药前。
推荐检测的适应症：1、少精子症患者（精子数目少于20×106/ml）；2、精子密度正常，但原因不明的男性不育症患者；3、男性不育伴隐睾和精索静脉曲张的患者；4、妻子有不明原因习惯性流产的患者。
诊断实验指南Y染色体上存在影响精子发生的无精子因子（AZF）区域，进一步可分为AZFa,AZFb和AZFc三个区域。
Y染色体微缺失分子诊断实验利用多重聚合酶链反应（multiplex－PCR）特异性扩增Y染色体AZF区域的序列标签位点（STS），扩增产物用电泳或杂交等方法进行检测。
中国人Y染色体微缺失分子诊断指南(草案)
中国人Y染色体微缺失分子诊断指南（草案）2005.4 上海前言在精子发生障碍引起的男性不育患者中，Y染色体微缺失的发生率仅次于Klinefelter’s syndrome(克氏综合征)，是居于第二位的遗传因素。
Y染色体微缺失已成为男性不育患者的常规检查项目。
欧洲男科学协会和欧洲分子遗传实验质控协作网为提高诊断质量，在1999年和2004年先后发布了第一版和第二版Y染色体微缺失分子诊断指南。
经过多年的临床实践证明该指南准确、灵敏和易于操作。
2005年4月在上海召开了中国人Y 染色体微缺失分子诊断的研讨会，成立了Y染色体微缺失分子诊断协作网。
会议在欧洲指南的基础上起草了符合目前我国男性不育诊疗现状，并反应最新生物技术发展的中国人Y染色体微缺失分子诊断指南。
本指南重点讨论Y染色体微缺失分子诊断具体实施时的标准化和规范化，推荐的相关方法和设计是根据欧洲指南和我国已有的实验研究基础上综合而成。
对机理研究和背景知识介绍部分在本指南中不再详细讨论。
男性不育症患者Y染色体微缺失分子筛查适应症常规检测的适应症：1、男性不育症患者选择单精子卵泡浆内注射（ICSI）或体外受精（IVF）生育子代前；2、非梗阻性无精子症患者；3、严重少精子症患者（精子数目少于5×106/ml）；4、无精子症患者进行睾丸活检术前；5、男性不育症患者（如精索静脉曲张）手术前；6、原因不明的男性不育症患者用药前。
推荐检测的适应症：1、少精子症患者（精子数目少于20×106/ml）；2、精子密度正常，但原因不明的男性不育症患者；3、男性不育伴隐睾和精索静脉曲张的患者；4、妻子有不明原因习惯性流产的患者。
诊断实验指南Y染色体上存在影响精子发生的无精子因子（AZF）区域，进一步可分为AZFa,AZFb和AZFc三个区域。
Y染色体微缺失分子诊断实验利用多重聚合酶链反应（multiplex－PCR）特异性扩增Y染色体AZF区域的序列标签位点（STS），扩增产物用电泳或杂交等方法进行检测。
Y染色体微缺失检测指导
Y染色体微缺失是严重少精子或无精子症的重要原因，是导致男性不育的第二大遗传因素，其发生率仅次于Klinefelter综合征(克氏综合征)。
从1999年开始，欧洲男科学协会和欧洲分子遗传实验质控网(EAA/EMQN)为提高诊断质量，出版了Y 染色体微缺失分子诊断指南，并提供了客观的实验质量评价方法。
最新版的实验室指南是2013年9月EAA/EMQN根据12年的临床积累和专家共识，在1999版和2004版的基础上修订而成。
新指南重点阐明：在少精子症或无精子症男性中。
测指南有重要的指导意义。
一、Y染色体微缺失发生频率Y染色体微缺失在健康人群中发生率约为1/4 000，但在不育男性中显着升高，微缺失发生频率为2%～10%(甚至更高)。
2013版指南指出Y染色体微缺失在中国不育男性中发生频率为11.5%，处于较高水平。
2006年朱晓斌等[1]针对中国不育男性染色体的研究表明AZF微缺失在非梗阻性无精子症患者中的发生率为9.94%，严重少精子症患者中发生率为5.82%，与国内外其他学者的研究基本一致。
随着微缺失分子机制的阐明和Y染色体男性特异区域的结构(MSY)测序完成，结合十多年临床数据分析，EAA专家总结沿用Repping等[2]对Y染色体微缺失区域的定义模式，分为：AZFa区缺失、AZFb区缺失、AZFbc区缺失和AZFc区缺失，认为只有该微缺失模式有明确的临床表现。
国内外学者对AZFd区缺失是否存在一直存有争议。
尽管发现在AZFb与AZFc两区域之间存在新的缺失位点(一些学者认为的AZFd区缺失)，但是该区域缺失没有明确的临床意义，也并非独立存在的缺失模式。
所以第四区域AZFd区缺失仍存在较多争议。
Müslümanolu等[3]在2005年的一项研究中指出AZFd缺失可能与精子形成有关，但仍缺乏有力的临床证据。
2013年，陈科等[4]在精子正常和轻度少精子症患者中都发现了假定的AZFd 区缺失。
浙中地区男性不育患者Y染色体微缺失检测分析
中国乡村医药浙中地区男性不育患者Y染色体微缺失检测分析张 恒 刘 颖 吴海啸 吴 汉 杨 庆 胡 洋 黄 汀不孕不育是影响人类生殖健康的重要问题，其中导致男性不育的原因有多方面，如遗传因素、下丘脑-垂体病变、生殖器外伤、输精管梗阻等。
其中遗传因素是临床上导致男性不育的重要因素。
在总人群中Y染色体微缺失的发生率约为1/4000，但在无精或少精的男性中，其发生率明显提高，为2%～10%，有的地区甚至更高[1-2]。
为了解浙中地区Y染色体微缺失在无精或少精男性患者中的发生情况，笔者采用荧光PCR法对321例患者进行Y染色体微缺失检测。
1 资料与方法1.1 对象与分组 选取2017年1月至2019年5月我院就诊的无精子症患者89例为无精子组、严重少精子患者136例为严重少精子组、精液质量正常者96例为正常对照组，年龄22～45岁。
排除梗阻性无精患者，按照WHO静液分析标准，精子密度＜5×106/ml为严重少精。
1.2 荧光定量PCR法检测 采用乙二胺四乙酸抗凝真空采血管采集患者外周静脉血2ml，-20℃保存备用。
取200μL 抗凝血，采用新鲜血液DNA提取试剂盒（厦门艾德生物医药科技股份有限公司）提取基因组DNA，按照试剂盒说明书进行操作。
使用NanoDrop 2000微量紫外分光光度计进行DNA浓度检测。
Y染色体无精子因子（AZF）基因微缺作者单位：321000 浙江金华市中心医院泌尿外科（张恒、吴海啸、吴汉、杨庆、胡洋、黄汀），中心实验室（刘颖）通信作者，刘颖，Email:***********************失检测采用Y染色体微缺失检测试剂盒（厦门艾德生物医药科技股份有限公司）进行。
使用欧洲男科学协会/欧洲分子遗传质量协作网（EAA/EMQN）推荐的序列标签位点（STS），检测Y染色体AZFa、AZFb、AZFc三个区域的缺失状态，每个区域分别以两个STS位点为代表，AZFa区检测的STS位点为sY84和sY86，AZFb区检测的STS位点为sY127和sY134，AZFc区检测的STS位点为sY254和sY255。
哪些人需要筛查Y染色体微缺
哪些人需要筛查Y染色体微缺Y染色体微缺失如何产生？精子发生是一个简单的过程，且全部的过程都受基因的调控，估量有2000个基因调整精子的生成，绝大多数位于常染色体区，少数基因位于Y染色体上。
Y染色体不具有DNA的修复功能，且不参加生殖以外的其他生理功能调整。
由此认为Y染色体的缺失发生较其他染色体简单。
由于全部的Y染色体基因都是单倍体，故一个基因的缺失就能产生效应。
哪些因素引起Y染色体微缺失？有学者认为Y染色体缺失是一种随机大事，不依靠于Y染色体消失的背景，可能是其他的遗传或坏境因素所致。
关于详细的因素还不明确，但其与男性不育的相关性已经得到证明。
Y染色体微缺失的基因及表型Y染色体缺失发生的区域主要分为3个：AZFa、AZFb、AZFc，以AZFc 的缺失最多，约占60％。
Y染色体微缺失遗传型与表型的关系已进行了大量的讨论，对临床有特别好的指导意义。
AZFa，AZFb，AZFc三个区域全部缺失的患者，100％表现为无精子症。
不行能通过任何手段从睾丸中获得精子。
AZFa区域整段缺失通常导致唯支持细胞综合症（SCO综合症），临床表现为无精子症。
假如诊断为整段AZFa区域缺失，若想从睾丸中获得精子进行ICSI已不大可能。
AZFb和AZFb＋c整段缺失得典型睾丸组织学特征是SCO综合症或生精阻滞。
与AZFa区域整段缺失的状况类似，这种病人在睾丸穿刺时也找不到精子。
因此，不推举给这类病人实施ICSI。
AZFc缺失的临床和睾丸组织学表现多中多样。
一般说来，AZFc缺失患者尚存在精子生成力量。
AZFc缺失见于无精子症和严峻少精子症患者，罕见状况下，也可以在自然状况下遗传给男性后代。
在无精子症患者中，AZFc缺失者通过TESE获得精子的机会要大的多，也可进行ICSI受孕。
但这些患者的男性子代将是AZFc缺失的携带者。
另外，有讨论发觉AZFc区域缺失的少精子症患者，其精子数目有进行下降的趋势，最终进展为无精子症。
因此，对AZFc区域缺失的少精子患者，应及早进行治疗或其精液进行冷冻保存。
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丫染色体微缺失是严重少精子或无精子症的重要原因，是导致男性不育的第二大遗传因素，其发生率仅次于Klinefelter综合征（克氏综合征）。
从1999年开始，欧洲男科学协会和欧洲分子遗传实验质控网（EAA/EMQN为提高诊断质量，出版了丫染色体微缺失分子诊断指南，并提供了客观的实验质量评价方法。
最新版的实验室指南是2013年9月EAA/EMQN根据12年的临床积累和专家共识，在1999版和2004版的基础上修订而成。
新指南重点阐明：在少精子症或无精子症男性中发现的丫染色体微缺失区域，主要是无精子因子（azoospermia factor，AZF）区域仅包含AZFa AZFb AZFbc AZFc和AZFabc区，独立的AZFd区并不存在；AZFc 区中gr/gr 缺失是影响精子生成的一个危险因素，但临床意义尚存争议，不作为常规检查指标；检测位点增加并不能提高检测灵敏度，反而可能使结果复杂化；基于两管多重PCR的检测方法仍适用于整个AZF缺失检测。
EAA/EMQN 12年国际质量评估计划（EQA计划）的实施表明，参与实验室通过规范实验操作，改善报告质量，可有效降低诊断错误率。
丫染色体微缺失在中国不育男性中的发生频率为11.5%，处于较高水平，我们建议将AZF检测作为男性严重少精子或无精子症的常规检测项目，呼吁国内AZF检测实验室加入EQA计划，完善中国丫染色体微缺失检测实验操作规范。
丫染色体微缺失分子检测在中国已开展多年，国内专家对AZF缺失模式、检测序列标签位点（sequenee- tagged site, STS的数量、检测方法和内部质量控制等临床问题未达成共识。
各实验室的诊断操作方法有很大不同，导致不准确或错误诊断时有发生，迫切需要建立丫染色体微缺失诊断标准和质量控制标准。
EAA/EMQN 更新的2013版指南对以上问题都给出了明确的专家共识，对我国建立自己的检测指南有重要的指导意义。
一、丫染色体微缺失发生频率丫染色体微缺失在健康人群中发生率约为1/4 000，但在不育男性中显着升高，微缺失发生频率为2%〜10%（甚至更高）。
2013版指南指出丫染色体微缺失在中国不育男性中发生频率为11.5%,处于较高水平。
2006年朱晓斌等［1］针对中国不育男性染色体的研究表明AZF微缺失在非梗阻性无精子症患者中的发生率为9.94%，严重少精子症患者中发生率为5.82%，与国内外其他学者的研究基本一致。
随着微缺失分子机制的阐明和丫染色体男性特异区域的结构（MSY）测序完成，结合十多年临床数据分析，EAA专家总结沿用Repping等［2］对丫染色体微缺失区域的定义模式，分为：AZFa区缺失、AZFb区缺失、AZFbc区缺失和AZFc区缺失，认为只有该微缺失模式有明确的临床表现。
国内外学者对AZFd区缺失是否存在一直存有争议。
尽管发现在AZFb与AZFc两区域之间存在新的缺失位点（一些学者认为的AZFd区缺失），但是该区域缺失没有明确的临床意义，也并非独立存在的缺失模式。
所以第四区域AZFd区缺失仍存在较多争议。
M tsltmanolu等［3］在2005年的一项研究中指出AZFd缺失可能与精子形成有关，但仍缺乏有力的临床证据。
2013年，陈科等［4］在精子正常和轻度少精子症患者中都发现了假定的AZFd 区缺失。
然而，至今为止AZFd区域尚未发现与精子生成相关的候选基因，其缺失所对应的临床表型还需进一步确认。
二、gr/gr缺失不纳入常规检查项目研究证实gr/gr缺失属于AZFc部分缺失，是影响精子生成的一个重要因素，但是否需要将其作为常规检查指标尚存争议。
尽管gr/gr缺失可导致AZFc区一半以上基因丢失，但其临床意义仍不明确，因为该缺失患者精子生成表型变化多样，从少精到精子数目正常都存在。
gr/gr缺失表型在不同人种和地域间存在差异，已有研究证实在欧洲白种人（除了法国人⑸和德国人［6］）和西太平洋居民中，gr/gr 缺失是男性生精障碍的高危因素［7］。
但在部分亚洲国家如日本和中国［8,9］,gr/gr 缺失在健康人群中的概率和在不育患者的概率无显着差别。
李铮等［10］研究发现Y染色体gr/gr缺失既存在于严重不育男性，也存在于生精功能正常的捐精者中，缺失可能遗传自父亲，并未影响精子的发生，不能认为是精子发生的遗传风险因子。
因此在中国gr/gr缺失不建议作为丫染色体微缺失常规检测的缺失模式。
三、AZF缺失基因型/表型相关性丫染色体微缺失主要是AZF的缺失，该区域包含精子生成的相关基因家族，不同程度的缺失可导致少精子或无精子症。
AZF区进一步可分为AZFa AZFb AZFc区域。
AZFa区域缺失通常导致唯支持细胞综合征（SCO综合征）和无精子症。
如果诊断为整个AZFa区域缺失，从睾丸中获得精子的概率为0。
AZFb和AZFbc（P5/P1 近端；P5/P1远端，P4/P1远端）缺失的典型睾丸组织学特征是SCO综合征或精子发生阻滞导致的无精子症。
研究表明这些缺失与整个AZFa区域缺失情况一样，患者也不能通过睾丸穿刺（TESE获得精子。
严重少精子或无精子症患者中AZFc 缺失发生频率最高，AZFc缺失的临床表型和睾丸组织学类型多种多样。
一般说来，AZFc 缺失患者尚残存精子生成能力。
罕见情况下，其缺失在自然状态下可遗传给其男性后代［11］。
近50%AZFc缺失的无精子症患者可通过TESE获得精子，进行单精子卵胞浆内注射（ICSI受孕，但这些患者的男性后代都将是AZFc缺失的携带者。
四、丫染色体微缺失检测人群丫染色体微缺失检测不仅可以明确严重少精子或无精子症的病因，而且可以对预后做出一定的评估。
丫染色体微缺失通常见于无精子症或精子浓度少于 2 X106/ml的患者。
一般的临床参数，如激素水平、睾丸大小、精索静脉曲张、睾丸畸形、感染等对是否存在丫染色体微缺失没有任何预测价值，丫染色体微缺失必须通过分子检测来确定。
拟行ICS的严重少精子症应该做丫染色体微缺失检测，而对非梗阻性无精子症患者在行TESE或者睾丸显微切开取精术前也应该进行常规检测，因为当整个AZFa整个AZFb AZFbc或者AZFabc缺失时都不建议给患者做手术。
需要特别提醒的是，如果采用辅助生育技术，AZFc缺失可以垂直遗传给男性后代。
如果患者通过AZF检测发现部分AZFa AZFb或AZFc缺失，建议家族中其他男性也进行AZF检测，因为这种缺失是可以遗传的。
但整个AZFa AZFb AZFbc或者AZFabc缺失时不建议家族其他男性成员做AZF检测，因为这些缺失通常没有精子生成。
对丈夫携带AZFc区缺失类型的夫妻，研究其ICSI受孕情况［12］，大部分研究表明Y染色体微缺失存在与否不影响受精率和受孕率，但也有个别报道指出微缺失会导致受精率下降，影响胚胎质量，降低囊胚率和ICSI成功率［13］。
五、STS丫染色体微缺失上的STS位点高达131个，如果用于丫染色体微缺失检测的STS 过少，将有可能漏检某些区域的缺失，但若采用的STS过多，则可能包含一些多态性序列，而这些位点在正常可育男性中也可出现缺失。
指南指出，原则上只要对每个AZF区域一个非多态性的STS位点进行分析就足以判断AZFa AZFb AZFc是否存在缺失。
任何一个已知缺失区域都包括多个STS位点，每个区域设置2个STS位点有助于增加检测的准确性。
鉴于众多实验室经验总结和外部质量控制，并考虑到多重PCR T增的形式，在1999和2004版指南中推荐的经典STS引物仍然有效，这些引物包括：AZFa: sY84sY86;AZFbsY127 SY134; AZFc: sY254 sY255都在DAZ基因上）。
这些STS位点引物由多个实验室和外部质量控制实验证明：结果可信重复性好。
采用这些引物几乎能够检测到所有临床上的相关缺失和文献报道的3个AZF区域95%以上的缺失，设计的这套引物能完全满足常规检测。
它是一个简单的标准，便于实验室质量控制和缩小实验室之间的检测差异，因此强烈推荐所有实验室都采用这些引物。
指南指出一些检测方法和试剂可检测很多STS位点，但并不能提高检测的灵敏度，反而可能使结果复杂化、难以解释，因为这可能检测到很多假的缺失位点，尤其是当DNA质量不好，PCR条件不是最佳时。
而大部分试剂盒并没有额外的单重PCR来确认可疑结果，因此指南并不推荐增加STS位点［14］。
六、PCR的形式和内部质量控制丫染色体微缺失检测采用多重PCR体系，体系中设置的PCR引物应该包括2个内对照：sY14（SRY） ZFX/ZFY 6 个STS位点：sY84 sY86 sY127 sY134 sY254 sY255 PCR需设立内对照（SRY ZFX/ZFY S因），阳性对照（健康的男性DNA）,阴性对照（女性DNA）和水空白对照（用水代替模板，即含有除模板DNA外所有成分的PCR反应）。
阳性对照监测实验操作是否有效，阴性对照监测DNA是否污染，空白对照监测试剂是否污染。
内对照SRY基因是男性性别决定基因，位于Y染色体短臂，内对照SRY S因可以在ZFY基因丢失时证明丫染色体特征序列的存在（比如:在XX男性中）。
ZFX基因检测不仅可以作为女性DNA对照，也可作为没有SRY 基因的46, XX男性患者唯一的阳性对照。
指南推荐的相关操作都经过认真设计和优化，采用两管多重PCR反应，每管多重PCR反应体系中都包括每个区域的一个位点。
当标本出现整个AZFa AZFb或AZFc 区缺失时，两管PCR反应中相应区域设置的2个STS位点产物都会缺失。
当AZFa 和AZFb区域出现部分缺失时，相关区域单个位点的缺失是有可能的，但需小心求证，对整个区域进行详细的研究，目前这种情况很少见，被认为是例外。
通常认为AZFc区的sY254或sY255单个缺失是实验结果错误。
七、丫染色体微缺失检测方法EAA/EMQN推荐采用多重PCR方法检测丫染色体微缺失。
基于多重PCR的丫染色体微缺失检测方法很多，如实时荧光PCR（Real time PCR）电泳法和芯片法等。
Rea- time PCR检测采用荧光标记探针，不涉及电泳，检测速度更快，数字化结果更加直观，因此指南推荐有条件的实验室都应采用该方法。
在没有Real- time PCR检测仪的实验室，可采用电泳法或毛细电泳法。
其他的检测方法如基因芯片检测，其花费昂贵，且包含太多不必要的位点，不为指南所推荐。
实验室建立一种新的检测方法，都需经过大量样本验证，并明确指出所采用的方法，而不是简单的描述"根据指南方法"。
指南推荐方法特指两管多重PCR方法，检测位点如上文所述6个经典位点。
八、EAA/EMQN 12年实验总结进行AZF检测的实验室应该加入"EQA计划"。