A novel universal multiplex PCR improves detection of AZFc Y-chromosome microdeletions

  • Hong-Yun Zheng
  • Yan Li
  • Fu-Jin Shen
  • Yong-Qing Tong
Technological Innovations



To determine the frequencies and the characteristics of Y chromosome microdeletions (pl) in infertile men from central China to perform appropriate therapeutic choices by updated multiplex-PCR.


In this study, we established a novel universal primer-multiplex-PCR (U-M-PCR) method to overcome the disadvantages of traditional multiplex PCR (M-PCR). We chose 15 sequence-tagged sites (STS) for detection of Y chromosome microdeletions. 540 infertile male patients and 100 healthy male controls were selected in the study.


Of the 540 male infertility patients, 48 Y-chromosome microdeletions were detected, with a total deletion rate of 8.9 %. Of these deletions, the rate of AZFa deletions (sY84) was 0.5 % (3/540), the rate of AZFb deletions (sY143) was 0.7 % (4/540) and the rate of AZFc deletions (sY242, sY254 and sY255) was 7.6 % (41/540). Compared with AZF deletion rates by M-PCR, we found U-M-PCR could detect AZFc deletion more specifically (1.0 % & 7.6 %). No Y-chromosome microdeletions were detected in the 100 males with normal semen (the control group).


U-M-PCR method was more specific to detect AZFc microdeletions. It is necessary to use the U-M-PCR method to offer genetic screening and counseling to infertile men prior to intracytoplasmic sperm injection (ICSI) or in-vitro fertilization (IVF).


Male infertility Y-chromosome Microdeletions Multiplex PCR AZFc 



This research supported by the forty-ninth issue of the China Postdoctoral Science Foundation, first-class funding (Grant No. 20110490119), National Natural Science Foundation of China (Grant No. 81100959) and National Clinical Key Specialty Construction Projects of the Health Ministry (2010).

Author contributions

ZHY carried out the multiplex PCR, performed the statistical analysis and drafted the manuscript. SFJ and TYQ collected the samples and extracted genomic DNA from peripheral blood. LY designed the study and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing financial interests

The authors have nothing to disclose.


  1. 1.
    Reijo R, Alagappan RK, Patrizio P, Page DC. Severe oligozoospermia resulting from deletions of azoospermia factor gene on Y chromosome. Lancet. 1996;347(9011):1290–3.PubMedCrossRefGoogle Scholar
  2. 2.
    Lange J, Skaletsky H, van Daalen SK, Embry SL, Korver CM, Brown LG, et al. Isodicentric Y chromosomes and sex disorders as byproducts of homologous recombination that maintains palindromes. Cell. 2009;138(5):855–69. doi: 10.1016/j.cell.2009.07.042.PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Efstratiou A, George RC. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulcerans. World Health Organization. Commun Dis Public Health/PHLS. 1999;2(4):250–7.Google Scholar
  4. 4.
    Repping S, Skaletsky H, Lange J, Silber S, Van Der Veen F, Oates RD, et al. Recombination between palindromes P5 and P1 on the human Y chromosome causes massive deletions and spermatogenic failure. Am J Hum Genet. 2002;71(4):906–22. doi: 10.1086/342928.PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Skaletsky H, Kuroda-Kawaguchi T, Minx PJ, Cordum HS, Hillier L, Brown LG, et al. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature. 2003;423(6942):825–37. doi: 10.1038/nature01722.PubMedCrossRefGoogle Scholar
  6. 6.
    Jungwirth A, Giwercman A, Tournaye H, Diemer T, Kopa Z, Dohle G, et al. European Association of Urology guidelines on male infertility: the 2012 update. Eur Urol. 2012;62(2):324–32. doi: 10.1016/j.eururo.2012.04.048.PubMedCrossRefGoogle Scholar
  7. 7.
    Muslumanoglu MH, Turgut M, Cilingir O, Can C, Ozyurek Y, Artan S. Role of the AZFd locus in spermatogenesis. Fertil Steril. 2005;84(2):519–22. doi: 10.1016/j.fertnstert.2005.02.024.PubMedCrossRefGoogle Scholar
  8. 8.
    Cai ZM. [Y chromosome microdeletion and male infertility: past, present and future]. Zhonghua nan ke xue. Natl J Androl. 2010;16(5):387–94.Google Scholar
  9. 9.
    Demir B, Arikan II, Bozdag G, Esinler I, Karakoc Sokmensuer L, Gunalp S. ICSI outcome of patients with severe oligospermia vs. non-obstructive azoospermia. Clin Exp Obstet Gynecol. 2012;39(2):141–3.PubMedGoogle Scholar
  10. 10.
    Patrat C, Bienvenu T, Janny L, Faure AK, Fauque P, Aknin-Seifer I, et al. Clinical data and parenthood of 63 infertile and Y-microdeleted men. Fertil Steril. 2010;93(3):822–32. doi: 10.1016/j.fertnstert.2008.10.033.PubMedCrossRefGoogle Scholar
  11. 11.
    Xu W, Zhai Z, Huang K, Zhang N, Yuan Y, Shang Y, et al. A novel universal primer-multiplex-PCR method with sequencing gel electrophoresis analysis. PLoS ONE. 2012;7(1):e22900. doi: 10.1371/journal.pone.0022900.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Silber SJ, Disteche CM. In: Pagon RA, Adam MP, Bird TD, Dolan CR, Fong CT, Stephens K, editors. Y chromosome infertility. Seattle: GeneReviews; 1993.Google Scholar
  13. 13.
    Sachdeva K, Saxena R, Majumdar A, Chadda S, Verma IC. Use of ethnicity-specific sequence tag site markers for Y chromosome microdeletion studies. Genet Test Mol Biomark. 2011;15(6):451–9. doi: 10.1089/gtmb.2010.0159.CrossRefGoogle Scholar
  14. 14.
    Pinar A, Bozdemir N, Kocagoz T, Alacam R. Rapid detection of bacterial atypical pneumonia agents by multiplex PCR. Cent Eur J Public Health. 2004;12(1):3–5.PubMedCrossRefGoogle Scholar
  15. 15.
    Ding C, Cantor CR. A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc Natl Acad Sci U S A. 2003;100(6):3059–64. doi: 10.1073/pnas.0630494100.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Tettelin H, Radune D, Kasif S, Khouri H, Salzberg SL. Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project. Genomics. 1999;62(3):500–7. doi: 10.1006/geno.1999.6048.PubMedCrossRefGoogle Scholar
  17. 17.
    Inagaki S, Yamamoto Y, Doi Y, Takata T, Ishikawa T, Imabayashi K, et al. A new 39-plex analysis method for SNPs including 15 blood group loci. Forensic Sci Int. 2004;144(1):45–57. doi: 10.1016/j.forsciint.2004.03.005.PubMedCrossRefGoogle Scholar
  18. 18.
    Puzuka A, Pronina N, Grinfelde I, Erenpreiss J, Lejins V, Bars J, et al. Y chromosome–a tool in infertility studies of Latvian population. Genetika. 2011;47(3):394–400.PubMedGoogle Scholar
  19. 19.
    Akin H, Onay H, Turker E, Ozkinay F. Primary male infertility in Izmir/Turkey: a cytogenetic and molecular study of 187 infertile Turkish patients. J Assist Reprod Genet. 2011;28(5):419–23.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Mirfakhraie R, Mirzajani F, Kalantar SM, Montazeri M, Salsabili N, Pourmand GR, et al. High prevalence of AZFb microdeletion in Iranian patients with idiopathic non-obstructive azoospermia. Indian J Med Res. 2010;132:265–70.PubMedGoogle Scholar
  21. 21.
    Ferlin A, Arredi B, Speltra E, Cazzadore C, Selice R, Garolla A, et al. Molecular and clinical characterization of Y chromosome microdeletions in infertile men: a 10-year experience in Italy. J Clin Endocrinol Metab. 2007;92(3):762–70.PubMedCrossRefGoogle Scholar
  22. 22.
    Fu J, Li L, Lu G. Relationship between microdeletion on Y chromosome and patients with idiopathic azoospermia and severe oligozoospermia in the Chinese. Chin Med J. 2002;115(1):72–5.PubMedGoogle Scholar
  23. 23.
    Aknin-Seifer IE, Touraine RL, Lejeune H, Laurent JL, Lauras B, Levy R. A simple, low cost and non-invasive method for screening Y-chromosome microdeletions in infertile men. Hum Reprod. 2003;18(2):257–61.PubMedCrossRefGoogle Scholar
  24. 24.
    Mirfakhraie R, Mirzajani F, Kalantar SM, Montazeri M, Salsabili N, Pourmand GR, et al. High prevalence of AZFb microdeletion in Iranian patients with idiopathic non-obstructive azoospermia. Indian J Med Res. 2011;132:265–70.Google Scholar
  25. 25.
    Fu L, Xiong DK, Ding XP, Li C, Zhang LY, Ding M, et al. Genetic screening for chromosomal abnormalities and Y chromosome microdeletions in Chinese infertile men. J Assist Reprod Genet. 2012;29(6):521–7. doi: 10.1007/s10815-012-9741-y.PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Bu Y, Huang H, Wu HP, Zhang XD, Zhou GH, Cui YX, et al. Direct multiplex-PCR from whole blood for rapid detection of Y chromosome microdeletions. Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi. Chin J Med Genet. 2008;25(4):406–9.Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Hong-Yun Zheng
    • 1
    • 2
  • Yan Li
    • 1
    • 2
  • Fu-Jin Shen
    • 3
  • Yong-Qing Tong
    • 1
    • 2
  1. 1.Department of Clinical LaboratoryRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China
  2. 2.Clinical Molecular Diagnostic CenterRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China
  3. 3.Department of GynecologyRenmin Hospital of Wuhan UniversityWuhanPeople’s Republic of China

Personalised recommendations