Detection Of Y Chromosome Microdeletions In Patients With Severe Oligozoospermia And Azoospermia

ChapterⅠ: Screening for Y chromosome microdeletions in idiopathicand nonidiopathic infertile men with varicocele and cryptorchidismBackground:It has been estimated that up to 10％of couples worldwide suffer frominfertility and in approximately half of these cases the cause is defectivespermatogenesis. Azoospermia and oligozoospermia of unknown origin,in which obstruction of the vas deferens, other obvious urological reasonsand klinefelter syndrome have been ruled out, may be due to theabnormality of genes crucial to spermatogenesis. In regard tospermatogenesis gene, particular attention has been given to the Yq11region. The Y chromosome comprises 2％of the genome and consists ofa short (Yp) and long arm (Yq). 1976, Tiepolo and Zuffardi frist reported,from microscopic analysis of the Y chromosome in infertile men, that thehuman Y chromosome played crucial role in spermatogenesis.Cytogenetic and molecular studies of azoospermic and oligozoospermicmales have suggested the presence of azoospermia factors (AZF) in thehuman Y chromosome. Through the use of molecular markers that havebeen mapped at density on the Y chromosome, numerousdeletion-mapping studies have defined at least three distinctnon-overlapping intervals each associated with variable degrees ofspermatogenic impairment. Three regions located on the long arm of the Y chromosome were named azoospermia factor (AZF)-a, -b, and -c.Some authors have recently proposed the existence of a fourth region,AZFd, in closely proximity to AZFc. Deletion in four Y chromosomalregions-AZFa, AZFb, AZFc and AZFd -has been reported to disruptspermatogenesis and cause infertility. Several candidate genesresponsible for spermatogenesis have been identified in these regions andsome of them are thought to be functional in human spermatogenesis. In1993, Ma reported two cDNAs of a Y-specific gene family named theRNA binding motif (RBM) from a region on the long arm of the Ychromosome that was deleted in some infertiles patients. In 1995, DAZ(Deleted in azoospermia) was identified from analysis of AZFc intervalin the long arm of the Y chromosome in azoospermic men. AlthoughRBM and DAZ are not only gene present in this region, RBM and DAZgenes were isolated as candidate genes in controlling spermatogenesisfrom the AZFb and AZFc regions, respectively. Both of them encodeproteins that bind RNA and might be involved in its regulation andmetabolism.With the advances in assisted reproductive technology, microdeletionsof the Y chromosome will be transmitted to male offspring, and thesesons will be at risk of having the same infertility problem. Thispossibility should be conveyed to infertile couples with an explanation ofthe analysis of microdeletion of the Y chromosome.Object:This study aimed at assessing the frequency of microdeletions inChinese men with idiopathic and nonidiopathic infertility with varicoceleand cryptorchidism and discussing the clinical significance of the AZFregion.Material and Method:One hundred and forty-three azoospermia and severe oligozoospermia(2～5×10~6sperm/ml) were included in this study. We studied 40 age-matched healthy fertile men. Of the 143 subjects, 36 had a history ofunilateral cryptorchidism orchidopexied in childhood, 45 underwentvaricocelectomy because of varicocele, and 62 were classified asidiopathic patients. Semen samples were obtained on three differentoccasions, separated by a 3-week interval, following a 3-day period ofsexual abstinence, and complete semen analyses were performedaccording to WHO guidelines. Plasma concentrations offollicle-stimulating hormone (FSH) and testosterone (T) were determinedby radio immunoassay (RIA). All selected patients underwentexamination of the testes to evaluate testicular size. In all selectedpatients, idiopathic patients were studied with a comprehensive historyand general investigation to exclude other possible cause of testiculardamage, and varicocele or cryptorchidism was not associated with otherphenotypic anomalies. The testicular structure of infertile men wasanalyzed by means of bilateral fine needle aspiration cytology (FNAC).Genomic DNA was prepared from peripheral blood samples of theinfertile and fertile Chinese men, after informed consent had beenobtained, in order to screen for Y chromosome deletions by PCR. DNAswere stored at -20℃. Eight STS were analysed in two multiplex PCR:sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255for AZFc. The two multiplex PCR consisted of: Mix1: SRY(sY14)-ZFY-sY84-sY134-sY255 and Mix2: SRY (sY14)-ZFY-sY86-sY127-sY254. As internal controls, SRY and ZFY were included in bothPCR multiplexes. Several external controls are used for each PCRreaction: blank; female DNA; and fertile male DNA. The experimentswere done in triplicate. A STS was considered as absent only after 3amplification failures in the presence of successful amplification ofinternal control. To decrease the risk of contamination, a femaletechnician performed all experiments.Fisher's exact text was used to test the prevalence of Y chromosome microdeletions in three groups (idiopathic infertility, nonidiopathicinfertility and control group). Computations were performed with theSTATA statistical package version 7.0. P values less than 0.05 wereconsidered statistically significant.Result:1. Y-Chromosome microdeletions analysis: Successful amplification ofSRY and ZFY gene in patients confirmed good quality of DNA used inPCR reactions. Among normal karyotype infertile men, we found 21submicroscopic deletions (21/143, 14.7％). PCR analysis in 40 normalfertile men did not detect any abnormalities, while no amplification wasobserved in women and blank. PCR analysis with this set of Y-DNAmarkers showed deletions of portions of Yq in 12 of the 62 idiopathicpatients (12/62, 19.4％), and in 9 out of the 81 nonidiopathic patients(9/81, 11.1％). One patient had a microdeletions in the AZFa region, withsY84, sY86 (1/21, 4.8％); 2 patients presented with a large deletioninvolving sY127, sY143 from AZFb and sY254, sY255 from AZFc (2/21,9.5％); 2 deletions were located in a AZFb region (2/21, 9.5％); 16patients had a deletion in the AZFc region involving the DAZ (deleted inazoospermia) gene (16/21, 76.2％).2. Testicular histology of patients with micro-deletions: Our results oftestis biopsy samples from men with AZF deletions pointed to widehistological variations: testicular maturation arrest, severehypospermatogenesis, and sertoli cell-only syndrome have been found.Among 21 infertile men, four showed a testicular cytologic picture ofMA; six patients had SH; and 11 cases presented SCOS.3. Comparison of the frequency of Y chromosome microdeletions:Incidence of Y chromosome microdeletions involved in idiopathic,nonidiopathic and control group was 19.35％(12/62), 11.11％(9/81) and0.00％(0/40), respectively. There was no significant difference ofmicrodeletion incidences between idiopathic and nonidiopathic groups (P=0.233), but there was significant difference between idiopathic ornonidiopathic with control groups (P=0.003 and P=0.029, respectively).Conclusions:1. Our findings that Yq deletions can occur in 19.4％of malesdiagnosed as having idiopathic infertility and, significantly, in 11.1％ofnonidiopathic infertile males raise important questions concerning thescreening of Y deletions and genetic counseling. Current protocolsrecommend that men with idiopathic severe oligozoospermia ornonobstructive azoospermia be screened for Y chromosome deletions. Onthe basis of our data, we recommend a more extended screening programto include all men, idiopathic and nonidiopathic, seeking ICSI treatment.2. While the cause of zoospermia and severe oligozoospermia isunclear in most patients, we detected AZF microdeletions on the Ychromosome in some patients (14.7％). There may be other geneticfactors or unknown spermatogenesis impairment, such as the DAZLAgene mapped to chromosome 3, which may be the autosomal recessivegene responsible for male infertility. Accordingly, we must seek for newgenes related to spermatogenesis. ChapterⅡ: Comparative study on Y chromosome microdeletion ofidiopathic male infertility in peripheral leukocytes and testicularcellsBackground:Infertility is a major health problem today affecting about 10％ofcouples. Male factor is assumed to be responsible in about 50％of theinfertile couples. Various causative factors have been identified for maleinfertility, including varicocele, obstruction of spermatic ducts,agglutination of sperms, impotency, hormonal imbalance, and geneticdefects. Deletion mapping directed the discovery of genes related tospermatogenesis, and defined three regions as the azoospermia factors(AZFa, AZFb and AZFc) mapped to Yq11. These gene families areinvolved in pathogenic male infertility associated with azoospermia orsevere oligozoospermia. DFFRY (Drosophila Fat Facets Related Y) geneis currently considered as candidate gene for the AZFa phenotype. TheDFFRY gene might be involved in maintaining male germ cell lineage,like its X-homologue (DFFRX), which is involved in oocyte development.The main candidate in AZFb is the RBM (RNA-binding motif) genefamily whose expression is restricted to the testis. RBM consists ofapproximately 30 copies of genes and pseudogenes found on both armsof the Y chromosome, but it is suggested that functional genes are clustered at the Yq in the AZFb region. The main candidate gene in AZFcis the DAZ (Deleted in AZoospermia) cluster, a set of genes transcribedin the adult testis and expressed exclusively in germ cells. RBM andDAZ genes families encode RNA binding proteins with similar structuresrelated to hnRNPG family of proteins (heterogenous nuclearribonucleoproteins) involved in RNA metabolism, including packaging ofRNA, transporting to cytoplasm and splicing. The physical size of theseregions has been estimated to be 1-3 Mb for AZFa and AZFb and 3 Mbfor AZFc. Approximately 60％of the azoospermic and severeoligozoospermic men are defined as idiopathic. Studies indicate thatbetween 10-15％of men with idiopathic azoospermia and 5-10％of menwith oligozoospermia carry deletions of one or the other parts of the longarm of the Y chromosome. With the advances in artificial reproductivetechnology, microdeletions of the Y chromosome will be transmitted tomale offspring, and these sons will be at risk of having the sameinfertility problem. This possibility should be conveyed to infertilecouples with an explanation of the analysis of microdeletion of the Ychromosome.Object:In this study, we analyzed sample from Chinese men with idiopathicazoospermia or severe oligozoospermia using STS-PCR (SequenceTagged Sites-Polymerase Chain Reaction) and RT-PCR (ReverseTranscription- Polymerase Chain Reaction), in order to investigatewhether there was a difference on Y chromosome microdeletionsbetween peripheral leukocytes and testicular cells.Material and Method:1. Patient Selection and Clinical EvaluationSixty-two idiopathic patients were included in this study. Theinfertility group included 33 males with azoospermia and 29 with severeoligozoospermia (＜5×10~6sperm/ml). The age groups of these cases ranged from 25 to 39 years. Semen samples were obtained on threedifferent occasions, separated by a 3-week interval, following a 3-dayperiod of sexual abstinence, and complete semen analyses wereperformed according to WHO guidelines. Standard cytogenetictechniques of G banding were performed in each patient to exclude theones with chromosomal abnormalities. FSH (Plasma concentrations ofFollicle-stimulating hormone) and T (Testosterone) were determined byRIA (Radio immunoassay). All idiopathic patients were studied with acomprehensive history and general investigation to exclude other possiblecause of testicular damage, and varicocele or cryptorchidism was notassociated with other phenotypic anomalies. As comparison, we studied40 age-matched healthy fertile men and a female. Hospital EthicalCommittee approval and informed consent were obtained for all subjectsin this study.2. PCR Assay of Peripheral Blood SamplesGenomic DNA was prepared from peripheral blood samples of theinfertile and fertile Chinese men. DNA was stored at -20℃. Each patientwas tested for STS of AZF region located at long arm of Y-chromosome,including sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, sY254and sY255 for AZFc. The two multiplex PCR consisted of: Mix1: SRY(sY14)-ZFY-sY84-sY134-sY255 and Mix2: SRY (sY14)-ZFY-sY86-sY127-sY254. As internal controls, SRY (sex determining region) andZFY (Zinc finger of Y chromosome) were included in both PCRmultiplexes. Several external controls were used for each PCR reaction:blank; female DNA; and fertile male DNA. PCR was performed in a 50μL reaction volume containing 1.5mmol/L MgCl2, 10mmol/L Tris-HClpH8.0, 50mmol/L KCl, 200μmol/L each deoxyribonucleosidetriphosphate, 1 U Taq DNA polymerase, and 201μmol/L of each primer,sterile distilled water 50μL. After a preheating step at 95℃for 5 min, thePCR was performed with denaturing at 95℃for 1min, annealing at 56℃ for 30 sec, and primer extension at 72℃for 30 sec, in 35 cycles, withfinal incubation at 72℃for 5 min. Amplified products were separated on1.8％agarose gels. The experiments were done in triplicate. A STS wasconsidered absent only after 3 amplification failures in the presence ofsuccessful amplification of internal control. To decrease the risk ofcontamination, a female technician performed all experiments.3. RT-PCR Assay of Testicular CellsThe testicular material of infertile men was obtained by means ofFNAC (bilateral fine needle aspiration cytology). Briefly, bilateral fineneedle aspiration was performed using 23-gauge (0.6mm) butterflyneedles and aspirating with a 20 ml syringe. The 62 testicular tissueswere frozen in liquid nitrogen immediately after dissection andcryopreserved until the RNA was isolated. Total cellular RNA wasextracted from the testicular tissue using a Trizol-method. RT of totalRNA was performed using 0.2μg oligo-dT primer for 1 h at 42℃utilizing 25 U of avian myeloblastosis virus (AMV) reverse transcriptasein Tris 50 mmol/l(PH 8.3), KCL 75 mmol/1, MgCl23mmol/l,dithiothreitol(DTT)10 mmol/l, RNasin 1U (Promega, USA)and 1mmol/l of dATP, dGTP, dCTP, dTTP in a total volume of 20μl. Eachpatient was tested for STS of AZF region located at long arm ofY-chromosome, including DFFRY for AZFa, RBM for AZFb, sY254(DAZ) for AZFc. The PCR consisted of: SRY(sY14)-DFFRY-RBM-DAZ (sY254)-β-actin. As internal controls, SRYwas included in PCR experiment. The expression level ofβ-actin wasused as a reference to adjust for an equal amount of sample RNA. Briefly,PCR was carried out for each gene in 20μl of reaction volume containing:6μl of testicular cDNA diluted 1:20 from total cDNAobtained,Taqpolymerase (0.8U), dNTP (0.2mM dTTP, dCTP, dGTP, dATP),oligonucleotide primers(10 pmol each) made up in a final concentrationof 1×PCR reaction buffer(1.5 mmol/L MgCl2, 10mmol/L Tris-HCl pH8.3 and 50mmol/L KCl)。Amplification was performed for 35sequential cycles,each of them including 1 min of denaturation at 94℃, 1rain of primer annealing at 60℃and 1 min of extension at 72℃;beforethe first cycle, all samples were incubated for 10 min at 94℃. PCRreaction products were eventually stored at 4℃. Each PCR product wasdissolved using 1.5％agarose gel electrophoresis. All samples andstandards were amplified in triplicate.Result:1. Y- Chromosome microdeletions analysis of Peripheral BloodSamplesSuccessful amplification of SRY and ZFY gene in patients confirmsgood quality of DNA used in PCR reactions. Among normal karyotypeinfertile men, we found 12 submicroscopic deletions (12/62, 19.4％).Nine patients (9/33, 27.2％) were detected in the azoospermic group andthree (3/29, 10.3％) in the severe oligozoospermic group. PCR analysisin 40 normal fertile men did not detect any abnormalities, while noamplification was observed in women and blank. The results of thescreening are shown in Table 3 and Figure 1. It showed that 1 patients(subject 63) presented with a large deletion involving sY127, sY143 fromAZFb and sY254, sY255 from AZFc (1/21, 4.8％); 2 deletions (subject80 and 82) were located in a AZFb region (2/21, 9.5％); 9 patients(subject 9, 11, 27, 43, 69, 75, 77, 96 and 116) had a deletion in the AZFcregion involving the DAZ gene (9/21, 42.9％). Testosterone levels werewithin the normal range(7.4～52.4IU/L) in the 12patients with deletions,the mean being 33.3±1.5 IU/L. Of 12 patients, 7 had high FSH plasmaconcentrations (＞12.0IU/L, normal range 0.7～11.1IU/L); however, themean FSH level among patients with deletions (12.3±4.3 IU/L) did notdiffer significantly from the mean FSH level in patients without deletion(12.1±9.1 IU/L). Our results of testis biopsy samples from men withAZF deletions pointed to wide histological variations: SH (severe hypospermatogenesis), MA (maturation arrest), and SCOS (sertolicell-only syndrome) have been found. Among 12 infertile men, 3 showeda testicular cytologic picture of MA; 5 patients had SH; and 4 casespresented SCOS. Incidence of Y chromosome microdeletions involved inidiopathic and control group was 19.35％(12/62) and 0.00％(0/40).There was significant difference between idiopathic with control groups(p=0.003).2. Analysis of AZF gene expressed in testicular cellsRT-PCR was performed on RNA extracted from 62 testicular samplesusing the primer sets, DFFRY, RBM, DAZ and two control genes, SRYandβ-actin. The results of the screening are shown in Table 3 and Figure2. RT-PCR analysis from testicular cells gave normal amplifications forSRY and DFFRY mRNA in 62 idiopathic patients; two patients (subject80 and 82) were negative for RBM expression; No RBM and DAZ weredetected for subject 63; 9 patients (subject 9, 11, 27, 43, 69, 75, 77, 96and 116) had no expression in the AZFc region involving the DAZ gene.Of 9 cases, three patients (subject 28, 33 and 72) with normal PCRanalysis of DAZ gene on genomic DNA showed no RT-PCRamplification for DAZ mRNA. These results, together with normalamplification ofβ-actin from each samples allowed us to consider thevalidity of our experimental conditions.Conclusions:In conclusion, our results preliminary show that Yq microdeletiondetection is possible in testicular material. This approach may be used toanswer some current questions concerning the consequence of Ychromosome molecular technique at the gonadal level. Hence RT-PCRanalysis can also be helpful to define etiology of idiopathic infertility.Furthermore, ICSI (Intracytoplasmic sperm injection) is the mostsignificant recent development in treatment of male infertility, enablingcouples who were previously deemed infertile to produce offspring, however with the risk of passing on genetic abnormalities, and possiblydecreased fertility. In fact, deletions that occur in the germ line of thetestis would result in sperm that carry the deletion and, if these spermfertilized an oocyte, would give rise to an individual with a Ychromosomal deletion. Those men inherit the deleted chromosome fromthe germ line of their fathers. Thus, the combination of RT-PCRevaluation of specific spermatids expressed genes with PCR bloodevaluation might raise the odds for successful TESE (Testicular spermaspiration)-ICSI procedures, avoiding the similar genetic defectstransmission to offspring.