Mutation analysis in patients with total sperm immotility

  • Rute Pereira
  • Jorge Oliveira
  • Luis Ferraz
  • Alberto Barros
  • Rosário Santos
  • Mário Sousa



Perform the genetic characterization of five patients with total sperm immotility using Sanger sequencing and Whole Exome Sequencing (WES), in order to increase the knowledge on the genetics of sperm immotility and, ultimately, allow the identification of potential genetic markers for infertility.


Prospective study at a University Medical school. We analysed five men with total sperm immotility, four with dysplasia of the fibrous sheath (DFS), associated with disruption of several axonemal structures, and one patient with situs inversus totalis, which showed absence of dynein arms (DA) and nexin bridges. We screened 7 genes by Sanger sequencing, involved in sperm motility and associated to ultrastructural defects found in these patients (CCDC39, CCDC40, DNAH5, DNAI1, RSPH1, AKAP3 and AKAP4). Additionally, we performed WES analysis in the patient with situs inversus.


We identified nine new DNA sequence variants by WES. Two of these variants were considered particularly relevant: a homozygous missense change in CCDC103 gene (c.104G > C, p.R35P) probably related with absence of dynein arms; the other in the INSL6 gene (c.262_263delCC) is thought to be also involved in sperm immotility.


Our work suggests that WES is an effective strategy, especially as compared with conventional sequencing, to study highly heterogenic genetic diseases, such as sperm immotility. For future work we expect to expand the analysis of WES to the other four patients and complement findings with expression analysis or functional studies to determine the impact of the novel variants.


Dysplasia of the fibrous sheath (DFS) Genetic diagnosis situs inversus totalis Sperm immotility Whole Exome Sequencing (WES) 



We would like to acknowledge: Helena Oliveira, MSc, ESHRE Senior clinical embryologist; Ilda Pires, MSc, ESHRE Senior clinical embryologist and Madalena Cabral, BSc, Embryologist, for semen processing at CHVNG; Ana Gonçalves, BSc, and Cláudia Osório, BSc, for semen processing at CGR; Elsa Oliveira, 1st Class Technical Specialist of Pathology, Cytology and Thanatology in the Area of Diagnosis and Therapy and Ângela Alves, Technical assistant teaching and research, for semen processing for electron microscopy at ICBAS-UP.

We also would like to acknowledge Conceição Egas, PhD and Hugo Froufe, MSc (GenoInseq) for performing exome sequencing and assisting the initial variant filtering/analysis.


This work was financed by the Institutions of the authors and in part by UMIB, which is funded by National Funds through FCT-Foundation for Science and Technology, under the Pest-OE/SAU/UI0215/2014.

Disclosure statement

The authors have nothing to declare.

Supplementary material

10815_2015_474_MOESM1_ESM.docx (31 kb)
Supplementary Data 1 (DOCX 30 kb)
10815_2015_474_MOESM2_ESM.docx (36 kb)
Supplementary Figure 1 (DOCX 36 kb)
10815_2015_474_MOESM3_ESM.docx (148 kb)
Supplementary Figure 2 (DOCX 147 kb)


  1. 1.
    Curry MR, Watson PF. Sperm structure and function. In: Grudzinskas JG, Yovich JL, editors. Gametes - the spermatozoon. NY: Cambridge University Press; 1995. p. 45–69.Google Scholar
  2. 2.
    Eddy EM, Toshimori K, O’Brien DA. Fibrous sheath of mammalian spermatozoa. Microsc Res Tech. 2003;61:103–15.PubMedCrossRefGoogle Scholar
  3. 3.
    Burgess SA, Knight PJ. Is the dynein motor a winch? Curr Opin Struct Biol. 2004;14:138–46.PubMedCrossRefGoogle Scholar
  4. 4.
    Smith EF, Yang P. The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil Cytoskeleton. 2004;57:8–17.PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Wirschell M, Olbrich H, Werner C, Tritschler D, Bower R, Sale W, et al. The nexin-dynein regulatory complex subunit DRC1 is essential for motile cilia function in algae and humans. Nat Genet. 2013;45:262–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Ortega C, Verheyen G, Raick D, Camus M, Devroey P, Tournaye H. Absolute asthenozoospermia and ICSI: what are the options? Hum Reprod Update. 2011;17(5):684–92.PubMedCrossRefGoogle Scholar
  7. 7.
    Afzelius BA. The immotile-cilia syndrome: a microtubule-associated defect. Crit Rev Biochem Mol Biol. 1985;19:63–87.CrossRefGoogle Scholar
  8. 8.
    Boon M, Jorissen M, Proesmans M, De Boeck K. Primary ciliary dyskinesia, an orphan disease. Eur J Pediatr. 2013;172:151–62.PubMedCrossRefGoogle Scholar
  9. 9.
    Leigh MW, Pittman JE, Carson JL, Ferkol TW, Dell S, Davis SD, et al. Clinical and genetic aspects of primary ciliary dyskinesia/Kartagener syndrome. Genet Med. 2009;11:473–87.PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Pereira R, Oliveira J, Sousa M. A molecular approach to sperm immotility in humans: a review. Med Reprod y Embriol Clín. 2014;01(01):15–25.Google Scholar
  11. 11.
    Djakow J, Svobodová T, Hrach K, Uhlík J, Cinek O, Pohunek P. Effectiveness of sequencing selected exons of DNAH5 and DNAI1 in diagnosis of primary ciliary dyskinesia. Pediatr Pulmonol. 2012;47:864–75.PubMedCrossRefGoogle Scholar
  12. 12.
    Merveille A-C, Davis EE, Becker-Heck A, Legendre M, Amirav I, Bataille G, et al. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nat Genet. 2011;43:72–8.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Becker-Heck A, Zohn IE, Okabe N, Pollock A, Lenhart KB, Sullivan-Brown J, et al. The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation. Nat Genet. 2011;43:79–84.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Antony D, Becker-Heck A, Zariwala MA, Schmidts M, Onoufriadis A, Forouhan M, et al. Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms. Hum Mutat. 2013;34:462–72.PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Kott E, Legendre M, Copin B, Papon J-F, Dastot-Le Moal F, Montantin G, et al. Loss-of-function mutations in rsph1cause primary ciliary dyskinesia with central-complex and radial-spoke defects. Am J Hum Genet. 2013;93:561–70.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Chemes HE, Olmedo SB, Carrere C, Oses R, Carizza C, Leisner M, et al. Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men. Hum Reprod. 1998;13:2521–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Chemes HE, Rawe VY. The making of abnormal spermatozoa: cellular and molecular mechanisms underlying pathological spermiogenesis. Cell Tissue Res. 2010;341:349–57.PubMedCrossRefGoogle Scholar
  18. 18.
    Luconi M, Cantini G, Baldi E, Forti G. Role of a-kinase anchoring proteins (AKAPs) in reproduction. Front Biosci. 2011;16:1315–30.CrossRefGoogle Scholar
  19. 19.
    Turner RMO, Johnson LR, Haig-Ladewig L, Gerton GL, Moss SB. An X-linked gene encodes a major human sperm fibrous sheath protein, hAKAP82: genomic organization, protein kinase a-rii binding, and distribution of the precursor in the sperm tail. J Biol Chem. 1998;273:32135–41.PubMedCrossRefGoogle Scholar
  20. 20.
    Mandal A, Naaby-Hansen S, Wolkowicz MJ, Klotz K, Shetty J, Retief JD, et al. FSP95, a testis-specific 95-kilodalton fibrous sheath antigen that undergoes tyrosine phosphorylation in capacitated human spermatozoa. Biol Reprod. 1999;61:1184–97.PubMedCrossRefGoogle Scholar
  21. 21.
    Baccetti B, Collodel G, Gambera L, Moretti E, Serafini F, Piomboni P. Fluorescence in situ hybridization and molecular studies in infertile men with dysplasia of the fibrous sheath. Fertil Steril. 2005;84:123–9.PubMedCrossRefGoogle Scholar
  22. 22.
    Baccetti B, Collodel G, Estenoz M, Manca D, Moretti E, Piomboni P. Gene deletions in an infertile man with sperm fibrous sheath dysplasia. Hum Reprod. 2005;20:2790–4.PubMedCrossRefGoogle Scholar
  23. 23.
    Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 2012;13:134.PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Gaspar P, Lopes P, Oliveira J, Santos R, Dalgleish R, Oliveira JL. Variobox: automatic detection and annotation of human genetic variants. Hum Mutat. 2014;35(2):202–7.Google Scholar
  26. 26.
    Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Ng PC, Henikoff S. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res. 2003;31:3812–4.PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Schwarz JM, Rödelsperger C, Schuelke M, Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations. Nat Methods. 2010;7:575–6.PubMedCrossRefGoogle Scholar
  29. 29.
    Desmet F-O, Hamroun D, Lalande M, Collod-Béroud G, Claustres M, Béroud C. Human splicing finder: an online bioinformatics tool to predict splicing signals. Nucleic Acids Res. 2009;37:e67.PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Oliveira J, Negrao L, Fineza I, Taipa R, Melo-Pires M, Fortuna AM, et al. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing. J Hum Genet. 2015. doi: 10.1038/jhg.2015.20.Google Scholar
  31. 31.
    Paila U, Chapman BA, Kirchner R, Quinlan AR. GEMINI: integrative exploration of genetic variation and genome annotations. PLoS Comput Biol. 2013;9, e1003153.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25:25–9.PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Ewing B, Hillier L, Wendl MC, Green P. Base-calling of automated sequencer traces usingPhred. I. Accuracy assessment. Genome Res. 1998;8:175–85.PubMedCrossRefGoogle Scholar
  34. 34.
    Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–94.PubMedCrossRefGoogle Scholar
  35. 35.
    Doggett NA, Xie G, Meincke LJ, Sutherland RD, Mundt MO, Berbari NS, et al. A 360-kb interchromosomal duplication of the human HYDIN locus. Genomics. 2006;88:762–71.PubMedCrossRefGoogle Scholar
  36. 36.
    Berg JS, Evans JP, Leigh MW, Omran H, Bizon C, Mane K, et al. Next generation massively parallel sequencing of targeted exomes to identify genetic mutations in primary ciliary dyskinesia: implications for application to clinical testing. Genet Med. 2011;13:218–29.PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, et al. STRING v9. 1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013;41:D808–15.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Hornef N, Olbrich H, Horvath J, Zariwala MA, Fliegauf M, Loges NT, et al. DNAH5 mutations are a common cause of primary ciliary dyskinesia with outer dynein arm defects. Am J Respir Crit Care Med. 2006;174:120–6.PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Schwabe GC, Hoffmann K, Loges NT, Birker D, Rossier C, De Santi MM, et al. Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations. Hum Mutat. 2008;29:289–98.PubMedCrossRefGoogle Scholar
  40. 40.
    Knowles MR, Leigh MW, Carson JL, Davis SD, Dell SD, Ferkol TW, et al. Mutations of DNAH11 in patients with primary ciliary dyskinesia with normal ciliary ultrastructure. Thorax. 2012;67:433–41.PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Miki K, Willis WD, Brown PR, Goulding EH, Fulcher KD, Eddy EM. Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility. Dev Biol. 2002;248:331–42.PubMedCrossRefGoogle Scholar
  42. 42.
    Turner RMO, Musse MP, Mandal A, Klotz KEN, Friederike C, Jayes L, et al. Molecular genetic analysis of two human sperm fibrous. J Androl. 2001;22:302–15.PubMedGoogle Scholar
  43. 43.
    Moretti E, Scapigliati G, Pascarelli NA, Baccetti B, Collodel G. Localization of AKAP4 and tubulin proteins in sperm with reduced motility. Asian J Androl. 2007;9:641–9.PubMedCrossRefGoogle Scholar
  44. 44.
    Colantonio JR, Vermot J, Wu D, Langenbacher AD, Fraser S, Chen J-N, et al. The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear. Nature. 2008;457:205–9.PubMedCrossRefGoogle Scholar
  45. 45.
    Yeh S-D, Chen Y-J, Chang ACY, Ray R, She B-R, Lee W-S, et al. Isolation and properties of Gas8, a growth arrest-specific gene regulated during male gametogenesis to produce a protein associated with the sperm motility apparatus. J Biol Chem. 2002;277:6311–7.PubMedCrossRefGoogle Scholar
  46. 46.
    Bekker JM, Colantonio JR, Stephens AD, Clarke WT, King SJ, Hill KL, et al. Direct interaction of Gas11 with microtubules: implications for the dynein regulatory complex. Cell Motil Cytoskeleton. 2007;64:461–73.PubMedCrossRefGoogle Scholar
  47. 47.
    Asai DJ, Koonce MP. The dynein heavy chain: structure, mechanics and evolution. Trends Cell Biol. 2001;11:196–202.PubMedCrossRefGoogle Scholar
  48. 48.
    Silvanovich A, Li M, Serr M, Mische S, Hays TS. The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding. Mol Biol Cell. 2003;14:1355–65.PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Zukas R, Chang AJ, Rice M, Springer AL. Structural analysis of flagellar axonemes from inner arm dynein knockdown strains of Trypanosoma brucei. Biocell. 2012;36:133–42.PubMedGoogle Scholar
  50. 50.
    Panizzi JR, Becker-heck A, Castleman VH, Al-mutairi D, Liu Y, Loges NT, et al. CCDC103 mutations cause primary ciliary dyskinesia by disrupting assembly of ciliary dynein arms. Nat Genet. 2012;44:714–9.PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Anand-Ivell R, Dai Y, Ivell R. Neohormones as biomarkers of reproductive health. Fertil Steril. 2013;99:1153–60.PubMedCrossRefGoogle Scholar
  52. 52.
    Lok S, Johnston DS, Conklin D, Lofton-Day CE, Adams RL, Jelmberg AC, et al. Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat. Biol Reprod. 2000;62:1593–9.PubMedCrossRefGoogle Scholar
  53. 53.
    Burnicka-Turek O, Shirneshan K, Paprotta I, Grzmil P, Meinhardt A, Engel W, et al. Inactivation of insulin-like factor 6 disrupts the progression of spermatogenesis at late meiotic prophase. Endocrinology. 2009;150:4348–57.PubMedCrossRefGoogle Scholar
  54. 54.
    Chen G-W, Luo X, Liu Y-L, Jiang Q, Qian X-M, Guo Z-Y. R171H missense mutation of INSL6 in a patient with spermatogenic failure. Eur J Med Genet. 2011;54:e455–7.PubMedCrossRefGoogle Scholar
  55. 55.
    Knowles MR, Leigh MW, Ostrowski LE, Huang L, Carson JL, Hazucha MJ, et al. Exome sequencing identifies mutations in CCDC114as a cause of primary ciliary dyskinesia. Am J Hum Genet. 2013;92:99–106.PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Onoufriadis A, Shoemark A, Munye MM, James CT, Schmidts M, Patel M, et al. Combined exome and whole-genome sequencing identifies mutations in ARMC4 as a cause of primary ciliary dyskinesia with defects in the outer dynein arm. J Med Genet BMJ Publishing Group Ltd; 2014;51:61–7.Google Scholar
  57. 57.
    Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, Nickerson DA, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet. 2011;12:745–55.PubMedCrossRefGoogle Scholar
  58. 58.
    Teves ME, Zhang Z, Costanzo RM, Henderson SC, Corwin FD, Zweit J, et al. Sperm-associated antigen-17 gene is essential for motile cilia function and neonatal survival. Am J Respir Cell Mol Biol. 2013;48:765–72.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Rute Pereira
    • 1
    • 2
  • Jorge Oliveira
    • 3
    • 4
  • Luis Ferraz
    • 5
  • Alberto Barros
    • 6
    • 7
  • Rosário Santos
    • 3
    • 4
  • Mário Sousa
    • 1
    • 3
  1. 1.Laboratory of Cell Biology, Department of Microscopy, Institute of Biomedical Sciences Abel Salazar (ICBAS)University of Porto (UP)PortoPortugal
  2. 2.Department of Biology, Faculty of SciencesUniversity of PortoPortoPortugal
  3. 3.Multidisciplinary Unit for Biomedical Research-UMIBICBAS-UPPortoPortugal
  4. 4.Molecular Genetics Unit, Centre of Medical Genetics Dr. Jacinto Magalhães (CGMJM)Hospital Centre of Porto (CHP)PortoPortugal
  5. 5.Department of Urology, Hospital Eduardo Santos SilvaHospital Centre of Vila Nova de Gaia (CHVNG)Vila Nova de GaiaPortugal
  6. 6.Centre of Reproductive Genetics Alberto Barros (CGR)PortoPortugal
  7. 7.Department of Genetics, Faculty of MedicineUniversity of PortoPortoPortugal

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