OBJECTIVE: To determine the patterns and the prevalence of microdeletions in the azoospermia factor subregions of the Y chromosome in Hong Kong Chinese men with severe male-factor infertility. DESIGN: Controlled clinical study. SETTING: Reproductive centre of a university teaching hospital, Hong Kong. PARTICIPANTS: Fifty-eight men with severe male-factor infertility who participated in the in vitro fertilisation programme from May 1998 through March 1999, and 46 male volunteers with proven fertility. MAIN OUTCOME MEASURES: Polymerase chain reaction analysis of DNA from peripheral blood lymphocytes using six loci spanning the AZFa, AZFb, and AZFc subregions of the Y chromosome. RESULTS. No microdeletions were detected in the fertile controls or in patients with obstructive azoospermia. Deletions within the AZFc subregion were found in 9% (4/44) of men with non-obstructive azoospermia or severe oligospermia. Neither AZFa nor AZFb deletions were detected in any participants. CONCLUSION: Deletions within the azoospermia factor subregions of the Y chromosome are associated with severe male-factor infertility in Hong Kong Chinese men.published_or_final_versio

Ho, PC

Lau, EY

Ng, EH

So, WW

Tse, JY

Yeung, WS

Hong Kong Medical Journal

HKU Scholars Hub

TitleDeletions within the azoospermia factor subregions of the Ychromosome in Hong Kong Chinese men with severe male-factor infertility: controlled clinical study.Author(s) Tse, JY; Yeung, WS; Lau, EY; Ng, EH; So, WW; Ho, PCCitation Hong Kong Medical Journal = Xianggang Yi Xue Za Zhi / HongKong Academy Of Medicine, 2000, v. 6 n. 2, p. 143-146Issued Date 2000URL http://hdl.handle.net/10722/53571Rights Hong Kong Medical Journal. Copyright © Hong Kong MedicalAssociation.HKMJ Vol 6 No 2 June 2000      143Y-chromosome microdeletions in infertile menORIGINAL ARTICLESIntroductionInfertility affects approximately 15% of all marriedcouples in the general population, and half of the casesare male-related.1,2 Little is known, however, about thegenetic basis of male infertility. Tiepolo and Zuffardi3reported that 0.5% of a group of 1160 infertile menhad macroscopic deletions of the distal long arm ofthe Y chromosome (Yq). They proposed the presenceof an azoospermic factor (AZF) in this region. Theputative genes were subsequently mapped to threeclose subregions within the Yq11.22-23 region, namedAZFa, AZFb, and AZFc.4,5 The AZFa subregion wasfound in the proximal portion of deletion interval 5;AZFb was found in the proximal end of deletioninterval 6, extending into the distal part of deletioninterval 5; and AZFc was found in the distal portionof deletion interval 6. By using the polymerase chainreaction (PCR) and constructing Y-chromosomesequence-tagged site (STS) maps, Y-chromosomemicrodeletions have been detected in the AZF regionof DNA from infertile men.6 This study used the samemethodology to detect Y-chromosome microdeletionsin Hong Kong Chinese men with severe male-factorinfertility.MethodsParticipantsInfertile men were recruited from the in vitro fertilisa-tion programme of the Department of Obstetrics andGynaecology at The University of Hong Kong fromMay 1998 through March 1999. The study was approvedby the university ethics committee, and informed con-sent was obtained from each participant. Fifty-eightDepartment of Obstetrics and Gynaecology, The University of HongKong, Queen Mary Hospital, Pokfulam, Hong KongJYM Tse, PhDWSB Yeung, PhDEYL Lau, PhDEHY Ng, MB, BS, FHKAM (Obstetrics and Gynaecology)WWK So, MB, BS, FHKAM (Obstetrics and Gynaecology)PC Ho, MD, FHKAM (Obstetrics and Gynaecology)Correspondence to: Dr JYM TseDeletions within the azoospermia factor subregions ofthe Y chromosome in Hong Kong Chinese men withsevere male-factor infertility: controlled clinical studyJYM Tse, WSB Yeung, EYL Lau, EHY Ng, WWK So, PC HoObjective. To determine the patterns and the prevalence of microdeletions in the azoospermia factor subregionsof the Y chromosome in Hong Kong Chinese men with severe male-factor infertility.Design. Controlled clinical study.Setting. Reproductive centre of a university teaching hospital, Hong Kong.Participants. Fifty-eight men with severe male-factor infertility who participated in the in vitro fertilisationprogramme from May 1998 through March 1999, and 46 male volunteers with proven fertility.Main outcome measures. Polymerase chain reaction analysis of DNA from peripheral blood lymphocytesusing six loci spanning the AZFa, AZFb, and AZFc subregions of the Y chromosome.Results. No microdeletions were detected in the fertile controls or in patients with obstructive azoospermia.Deletions within the AZFc subregion were found in 9% (4/44) of men with non-obstructive azoospermia orsevere oligospermia. Neither AZFa nor AZFb deletions were detected in any participants.Conclusion. Deletions within the azoospermia factor subregions of the Y chromosome are associated withsevere male-factor infertility in Hong Kong Chinese men.HKMJ 2000;6:143-6Key words: Chromosome deletion; Infertility, male; Polymerase chain reaction; Y chromosome/genetics144      HKMJ Vol 6 No 2 June 2000Tse et almales with severe oligospermia (<1 x 106 spermatozoaper millilitre of ejaculate [n=9]), non-obstructiveazoospermia (n=35), or obstructive azoospermia dueto congenital or acquired causes (n=14) were recruited.Semen analysis was performed according to the 1992World Health Organization guidelines.7 Forty-sixhealthy men of proven fertility were recruited as positivecontrols.Screening for Y-chromosome microdeletionsAll tests were performed on genomic DNA that wasextracted from peripheral blood lymphocytes byusing a commercially available DNA-isolation kitfor mammalian blood (Boehringer MannheimCorp., Indiana, United States). For each participant, Ychromosome–specific STSs that spanned the AZFa(sY84 and sY86), AZFb (sY127 and sY132), and AZFc(sY254 and sY255) subregions were used to amplifysix specific regions of the Y chromosome using PCR.These markers were concentrated in the subintervals5 and 6 of Yq11, in which microdeletions have beenreported. To prevent false-negative results, each PCRsample was co-amplified with one of the two follow-ing internal control primers: sY72, a control markerfor the presence of Y chromosome–specific DNA;or globin, a marker indicating the quality of the DNApreparation and successful PCR amplification. For eachPCR sample, water and female genomic DNA wereused as controls to verify that no cross-contaminationhad occurred.The PCR amplification comprised a total volumeof 25 µL, which contained 100 to 200 ng of humangenomic DNA; 1.5 to 2.0 mmol/L magnesium chlor-ide; deoxyribonucleoside 5'-triphosphates (200-400 µmol/L each of dTTP, dCTP, dGTP, and dATP);0.2 to 1.0 µmol/L primer; PCR buffer; and 1 U Taqpolymerase (Boehringer Mannheim Corp., Indiana,United States). Thermocycling consisted of initialdenaturation of 4 minutes at 94°C and 35 cycles ofincubation at 94°C for 30 seconds, at 49 to 50°Cfor 30 seconds, and at 72°C for 1 minute. The PCRreaction products were stored at 4°C, separated on 2%to 3% agarose gels, and visualised by staining thegel with ethidium bromide. A sample was consideredpositive for an STS marker when the PCR productof the expected size was present; it was considerednegative if a product of the expected size was notamplified after three PCR attempts.ResultsAmong the 58 infertile men examined, neither AZFanor AZFb microdeletions were detected by PCRanalysis in any DNA samples. Furthermore, no AZFdeletions were detected in any of the 14 patients withobstructive azoospermia (congenital or acquired).However, AZFc microdeletions corresponding tothe STSs sY254 and sY255 were detected in theDNA samples of four infertile men: three with non-obstructive azoospermia and one with severe oligo-spermia. They constituted 6.9% (4/58) of all theinfertile men investigated or 9.1% (4/44) of those withnon-obstructive azoospermia or severe oligospermia.All four patients had microdeletions in subinterval 6of Yq11, which included the region of the AZF candi-date gene, DAZ (deleted in azoospermia). The PCRresults of some patients with deletions are shown inthe Figure. None of the 46 men with proven fertilityshowed deletions at any of the STSs tested.DiscussionThe prevalence of Y-chromosome microdeletions inthis study population was 6.9%. In contrast to studiesof Caucasian populations, in which Y-chromosomemicrodeletions were observed in a small proportion(1%) of infertile men with obstructive azoospermia,8no such microdeletions were detected in this studyamong the men whose infertility had an obstructivecause. On the other hand, of the participants with non-obstructive azoospermia or severe oligospermia, 9.1%(4/44) had microdeletions (Fig, lanes 1 and 2). Thisfigure is slightly lower than the frequencies (10%-15%)quoted in studies of Caucasian populations9-11; how-ever, these studies examined more loci. The frequencyof microdeletions found in this study is similar to thatfound in a report from Taiwan (9%).12 Although bothstudies used similar loci to detect the presence of Y-chromosome microdeletions, the study from Taiwandetected six AZF deletions in six different patients:three with deletions within the AZFa region and theother three with deletions within the AZFc region. Themicrodeletions of the four patients in this study wereall within the AZFc region.Zheng et al13 recently reported the prevalence ofY-chromosome microdeletions in AZF subregions of13.3% (12/90) in azoospermic or oligospermic men.13The figure is higher than that found in this study, buttheir results were not confirmed by including an internalcontrol primer in their PCR assay; hence, the absenceof amplification might have been because of PCRfailure or poor DNA quality. Furthermore, they did notinclude an additional primer within the AZFc regionin their PCR assay. In this study, AZFc deletion wasconfirmed by performing triplicate PCR amplificationswith two sets of primers within the AZFc region.HKMJ Vol 6 No 2 June 2000      145Y-chromosome microdeletions in infertile menRecent studies support the concept of the geneticbasis of male infertility.14-16 The high prevalence ratesof AZFc deletions among the local population and inCaucasian populations suggest that this region of theY chromosome consists of one or more important genesthat are responsible for normal spermatogenesis. TheDAZ gene, which clusters at the AZFc region, is one ofthe most frequently deleted genes in patients with se-vere male-factor infertility.16,17 This gene is expressedspecifically in the testis and encodes an RNA-bindingprotein. Although its precise biological function is notyet known, the DAZ protein is believed to play a rolein male germ-cell development.The development of intracytoplasmic sperm injec-tion (ICSI) has allowed many males with severe male-factor infertility to father a child. This technique,however, cannot cure the underlying spermatogenicproblem, and it is possible that the genetic defect istransmitted from the father to his offspring by virtueof the ICSI. We have recently found evidence for thisprocess in one patient (Fig, lanes 1 and 2): the sameAZFc microdeletion was detected in the oligospermicfather and his ICSI-derived baby.18 Hence, a sperm-atozoon with a Yq deletion in the AZFc subregion canfertilise an oocyte and result in pregnancy by meansof assisted reproduction techniques. 18,19This is the first study to investigate the patternand prevalence of Y-chromosome deletions in AZFsubregions in Hong Kong Chinese men with severemale-factor infertility. The prevalence of micro-deletions in the local population needs to be con-firmed by screening more infertile men and usingmore STS markers. Although Y-chromosome micro-deletions occur in a subgroup of infertile men, rou-tinely screening microdeletions in all male patientsbefore ICSI treatment is an important prerequisite totheir appropriate counselling.20AcknowledgementThis study was supported by a research grant (CRCG10201949/31846/20900/301/01) from The Universityof Hong Kong.References1. Hargreave TB. Introduction. In: Hargreave TB, Soon TE, edi-tors. The management of male infertility. Singapore: PG Pub-lishing; 1990.2. Skakkebaek NE, Giwercman A, de Kretser D. Pathogenesisand management of male infertility. Lancet 1994;343:1473-9.3. Tiepolo L, Zuffardi O. Localization of factors controllingspermatogenesis in the nonfluorescent portion of the humanY chromosome long arm. Hum Genet 1976;34:119-24.Fig. Results from multiplex polymerase chain reaction analysisLanes 1, 2, 4, and 5 show a deletion of sY254; lane 1: man with severe oligospermia; lane 2: intracytoplasmic sperminjection–derived baby from the patient in lane 1; lanes 3-5: men with non-obstructive azoospermia; lanes 6-12: fertilecontrols; lanes 13 and 14: negative control samples of female DNA and water, respectively; MW = 100-bp molecularweight markers1 2 3 4 5 6 7 8 9 10 11 12 13 14 MWsY254 →(350 bp)sY72 →(100 bp)146      HKMJ Vol 6 No 2 June 2000Tse et al4. Kobayashi K, Mizuno K, Hida A, et al. PCR analysis of the Ychromosome long arm in azoospermic patients: evidence for asecond locus required for spermatogenesis. Hum Mol Genet1995;4:974.5. Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosomeazoospermia factors (AZF) mapped to different subregions inYq11. Hum Mol Genet 1996;5:933-43.6. Foote S, Volrath D, Hilton A, et al. The human Y chromosome:overlapping DNA clones spanning the euchromatic region.Science 1992;258:60-6.7. World Health Organization. WHO laboratory manual for theexamination of human semen and sperm-cervical mucus inter-action. 3rd ed. Cambridge: The Press Syndicate of the Univer-sity of Cambridge;1992.8. Pryor JL, Kent-First M, Muallem A, et al. Microdeletions inthe Y chromosome of infertile men. N Engl J Med 1997;336:534-9.9. Nagafuchi S, Namiki M, Nakahori Y, Kondoh N, Okuyama A,Nakagome Y. A minute deletion of the Y chromosome in menwith azoospermia. J Urol 1993;150:1155-7.10. Najmabadi H, Huang V, Yen P, et al. Substantial prevalenceof microdeletions of the Y-chromosome in infertile men withidiopathic azoospermia and oligozoospermia detected using asequence-tagged site-based mapping strategy. J Clin EndocrinolMetab 1996;81:1347-52.11. Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defectsin humans caused by Y-chromosome deletions encompassinga novel RNA-binding protein gene. Nat Genet 1995;10:383-93.12. Chang SY, Tsai MY. Detection of azoospermic factor genesin Chinese men with azoospermia or severe oligozoospermia.J Assist Reprod Genet 1999;16:259-62.13. Zheng Y, Zhou Z, Wang L, et al. Analysis of the DAZ and DAZhgenes in spermatogenesis [in Chinese]. Shengzhi Yixue Zazhi1998;7:71-4.14. Chandley A. Chromosome anomalies and Y-chromosomemicrodeletions as causal factors in male infertility. Hum Reprod1998;(13 Suppl 1):45-50.15. Hargreave TB, Ghosh C, Cooke H. Genetics of male infertil-ity. Mol Cell Endocrinol 1998;145:143-51.16. Kleiman SE, Yogev L, Gamzu R, et al. Genetic evaluation ofinfertile men. Hum Reprod 1999;14:33-8.17. Silber SJ, Alagappan R, Brown LG, et al. Y-chromosomedeletions in azoospermic and severely oligozoospermic menundergoing intracytoplasmic sperm injection after testicularsperm extraction. Hum Reprod 1998;13:3332-7.18. Tse JY, Yeung WS, Lau EY, et al. Transmission of the Y-chromosome microdeletion to a baby boy conceived afterintracytoplasmic sperm injection. Chin Med J (Engl). In press2000.19. Page DC, Silber S, Brown LG. Men with infertility caused byAZFc deletion can produce sons by intracytoplasmic sperminjection, but are likely to transmit the deletion and infertility.Hum Reprod 1999;14:1722-6.20. Krausz C, Quintana-Murci L, McElreavey K. Prognosticvalue of Y deletion analysis: what is the clinical value ofY chromosome microdeletion analysis? Hum Reprod 2000;15:1431-4.

Deletions within the azoospermia factor subregions of the Y chromosome in Hong Kong Chinese men with severe male-factor infertility: controlled clinical study.

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Deletions within the azoospermia factor subregions of the Y chromosome in Hong Kong Chinese men with severe male-factor infertility: controlled clinical study.

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