Skip to main content

Genome-wide association study identifies candidate markers related to lincRNAs associated with male infertility in the Greek population



Male infertility is currently one of the most common problems faced by couples worldwide. We performed a GWAS on Greek population and gathered statistically significant SNPs in order to investigate whether they lie within or near lncRNA regions.


The aim of this study was to investigate whether polymorphisms on or near lncRNAs affect interactions with miRNAs and can cause male infertility.

Materials and methods

In the present study, a GWAS was conducted, using samples from 159 individuals (83 normozoospermic individuals and 76 patients of known fertility issues). Standard procedures for quality controls and association testing were followed, based on case-control testing.


We detected six lncRNAs (LINC02231, LINC00347, LINC02134, NCRNA00157, LINC02493, Lnc-CASK-1) that are associated with male infertility through their interaction with miRNAs. Furthermore, we identified the genes targeted by those miRNAs and highlighted their functions in spermatogenesis and the fertilization process.

Discussion and conclusion

lncRNAs are involved in spermatogenesis through their interaction with miRNAs. Thus, their study is very important, and it may contribute to the understanding of the molecular mechanisms underlying male infertility.

This is a preview of subscription content, access via your institution.

Fig. 1

Data availability

All data are openly available in the form of supplementary materials.


  1. 1.

    Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring Assisted Reproductive Technology (ICMART) and the World Health Organization (WHO) revised glossary of ART terminology, 2009∗. Fertil Steril. 2009, 2009;92:1520–4.

  2. 2.

    Massart A, Lissens W, Tournaye H, Stouffs K. Genetic causes of spermatogenic failure. Asian J Androl. 2012;14:40–8.

    CAS  Article  Google Scholar 

  3. 3.

    Smith JF, Walsh TJ, Shindel AW, Turek PJ, Wing H, Pasch L, et al. Sexual, marital, and social impact of a man's perceived infertility diagnosis. J Sex Med. 2009;6:2505–15.

  4. 4.

    Winters BR, Walsh TJ. The epidemiology of male infertility. Urol Clin N Am. 2014;41:195–204.

    Article  Google Scholar 

  5. 5.

    Krausz C, Riera-Escamilla A. Genetics of male infertility. Nat Rev Urol. 2018;15:369–84.

    CAS  Article  Google Scholar 

  6. 6.

    Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37.

    Article  Google Scholar 

  7. 7.

    Gabrielsen JS, Tanrikut C. Chronic exposures and male fertility: the impacts of environment, diet, and drug use on spermatogenesis. Andrology. 2016;4:648–61.

    CAS  Article  Google Scholar 

  8. 8.

    Jungwirth A, Diemer T, Kopa Z, Krausz C, Tournaye H. European Association of Urology (EAU) guidelines on male infertility. Arnhem, The Netherlands: European Association of Urology; 2015.

    Google Scholar 

  9. 9.

    Khalil AM, Guttman M, Huarte M, Garber M, Raj A, Rivea Morales D, et al. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc Natl Acad Sci U S A. 2009;106:11667–72.

  10. 10.

    Guttman M, Amit I, Garber M, French C, Lin MF, Feldser D, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature. 2009, 2009;458:223–7.

  11. 11.

    He Y, Vogelstein B, Velculescu VE, Papadopoulos N, Kinzler KW. The antisense transcriptomes of human cells. Science. 2008;322:1855–7.

    CAS  Article  Google Scholar 

  12. 12.

    Beermann J, Piccoli MT, Viereck J, Thum T. Non-coding RNAs in development and disease: background, mechanisms, and therapeutic approaches. Physiol Rev. 2016;96:1297–325.

    CAS  Article  Google Scholar 

  13. 13.

    Mukherjee A, Koli S, Reddy KVR. Regulatory non-coding transcripts in spermatogenesis: shedding light on “dark matter.”. Andrology. 2014;2:360–9.

    CAS  Article  Google Scholar 

  14. 14.

    Hayashi K, Chuva de Sousa Lopes SM, Kaneda M, Tang F, Hajkova P, Lao K, et al. MicroRNA biogenesis is required for mouse primordial germ cell development and spermatogenesis. PLoS One. 2008;3:e1738.

  15. 15.

    Maatouk DM, Loveland KL, McManus MT, Moore K, Harfe BD. Dicer1 is required for differentiation of the mouse germ line. Biol Reprod. 2008;79:696–703.

    CAS  Article  Google Scholar 

  16. 16.

    Korhonen HM, Meikar O, Yadav RP, Papaioannou MD, Romero Y, Da Ros M, et al. Dicer is required for haploid male germ cell differentiation in mice. PLoS One. 2011;6:e24821.

  17. 17.

    Kostereva N, Hofmann MC. Regulation of the spermatogonial stem cell niche. Reprod Domest Anim. 2008;43:386–92.

    Article  Google Scholar 

  18. 18.

    Björk JK, Sandqvist A, Elsing AN, Kotaja N, Sistonen L. miR-18, a member of Oncomir-1, targets heat shock transcription factor 2 in spermatogenesis. Development. 2010;137:3177–84.

    Article  Google Scholar 

  19. 19.

    Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22:1775–89.

  20. 20.

    Ransohoff J, Wei Y, Khavari P. The functions and unique features of long intergenic non-coding RNA. Nat Rev Mol Cell Biol. 2018;19:143–57.

    CAS  Article  Google Scholar 

  21. 21.

    Chen X, Yan CC, Zhang X, You ZH. Long non-coding RNAs and complex diseases: from experimental results to computational models. Brief Bioinform. 2017;18:558–76.

    CAS  Google Scholar 

  22. 22.

    Cabili MN, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A, et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011;25:1915–27.

  23. 23.

    Wichman L, Somasundaram S, Breindel C, Valerio DM, McCarrey JR, Hodges CA, et al. Dynamic expression of long noncoding RNAs reveals their potential roles in spermatogenesis and fertility. Biol Reprod. 2017;97:313–23.

  24. 24.

    Xue M, Zhuo Y, Shan B. MicroRNAs, long noncoding RNAs, and their functions in human disease. Methods Mol Biol. 1617;2017:1–25.

    Google Scholar 

  25. 25.

    Yoon JH, Abdelmohsen K, Gorospe M. Functional interactions among microRNAs and long noncoding RNAs. Semin Cell Dev Biol. 2014;34:9–14.

    CAS  Article  Google Scholar 

  26. 26.

    Paraskevopoulou MD, Hatzigeorgiou AG. Analyzing MiRNA–LncRNA interactions. Methods Mol Biol. 2016;140:271–86.

    Article  Google Scholar 

  27. 27.

    Sun B, Liu C, Li H, Zhang L, Luo G, Liang S, et al. Research progress on the interactions between long non-coding RNAs and microRNAs in human cancer. Oncol Lett. 2020;19:595–605.

  28. 28.

    Dong Z, Zhang A, Liu S, Lu F, Guo Y, Zhang G, et al. Aberrant methylation-mediated silencing of lncRNA MEG3 functions as a ceRNA in esophageal cancer. Mol Cancer Res. 2017;15:800–10.

  29. 29.

    Lü M, Tian H, Cao YX, He X, Chen L, Song X, et al. Downregulation of miR-320a/383-sponge-like long non-coding RNA NLC1-C (narcolepsy candidate-region 1 genes) is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation. Cell Death Dis. 2015;6:e1960.

  30. 30.

    Panoutsopoulou K, Hatzikotoulas K, Xifara DK, Colonna V, Farmaki AE, Ritchie GR, et al. Genetic characterization of Greek population isolates reveals strong genetic drift at missense and trait-associated variants. Nat Commun. 2014;5:5345.

  31. 31.

    Weyrich A. Preparation of genomic DNA from mammalian sperm. Curr Protoc Mol Biol. 2012;98:2.13.1–3.

    Article  Google Scholar 

  32. 32.

    Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am J Hum Genet. 2007;81:559–75.

  33. 33.

    International Schizophrenia Consortium, Purcell SM, Wray NR, Stone JL, Visscher PM, O'Donovan MC, et al. Common polygenic variation contributes to risk of schizophrenia and bipolar disorder. Nature. 2009;460:748–52.

  34. 34.

    Dayem Ullah AZ, Oscanoa J, Wang J, Nagano A, Lemoine NR, Chelala C. SNPnexus: assessing the functional relevance of genetic variation to facilitate the promise of precision medicine. Nucleic Acids Res. 2018;46:W109–13.

    Article  Google Scholar 

  35. 35.

    Volders PJ, Anckaert J, Verheggen K, Nuytens J, Martens L, Mestdagh P, et al. LNCipedia 5: towards a reference set of human long non-coding RNAs. Nucleic Acids Res. 2019;47:D135–9.

  36. 36.

    Paraskevopoulou MD, Vlachos IS, Karagkouni D, Georgakilas G, Kanellos I, Vergoulis T, et al. DIANA-LncBase v2: indexing microRNA targets on non-coding transcripts. Nucleic Acids Res. 2016;44:D231–8.

  37. 37.

    Kozomara A, Birgaoanu M, Griffiths-Jones S. miRBase: from microRNA sequences to function. Nucleic Acids Res. 2019;47:D155–62.

    CAS  Article  Google Scholar 

  38. 38.

    Hamberg M, Backes C, Fehlmann T, Hart M, Meder B, Meese E, et al. MiRTargetLink--miRNAs, genes and interaction networks. Int J Mol Sci. 2016;17:564.

  39. 39.

    Paraskevopoulou MD, Georgakilas G, Kostoulas N, Vlachos IS, Vergoulis T, Reczko M, et al. DIANA-microT web server v5.0: service integration into miRNA functional analysis workflows. Nucleic Acids Res. 2013;41:W169–73.

  40. 40.

    Meunier J, Lemoine F, Soumillon M, Liechti A, Weier M, Guschanski K, et al. Birth and expression evolution of mammalian microRNA genes. Genome Res. 2013;23:34–45.

  41. 41.

    Zhang R, Peng Y, Wang W, Su B. Rapid evolution of an X-linked microRNA cluster in primates. Genome Res. 2007;17:612–7.

    CAS  Article  Google Scholar 

  42. 42.

    Li Y, Wang H, Wan F, Liu F, Liu J, Zhang N, et al. Deep sequencing analysis of small non-coding RNAs reveals the diversity of microRNAs and piRNAs in the human epididymis. Gene. 2012;497:330–5.

  43. 43.

    Neto L, Bach PV, Najari BB, Li PS, Goldstein M. Spermatogenesis in humans and its affecting factors. Semin Cell Dev Biol. 2016;59:10–26.

    Article  Google Scholar 

  44. 44.

    Wu W, Qin Y, Li Z, Dong J, Dai J, Lu C, et al. Genome-wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p. Hum Reprod. 2013;28:1827–36.

  45. 45.

    Wang C, Yang C, Chen X, Yao B, Yang C, Zhu C, et al. Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility. Clin Chem. 2011;57:1722–31.

    CAS  Article  Google Scholar 

  46. 46.

    Abu-Halima M, Hammadeh M, Backes C, Fischer U, Leidinger P, Lubbad AM, et al. Panel of five microRNAs as potential biomarkers for the diagnosis and assessment of male infertility. Fertil Steril. 2012;102:989–97.

  47. 47.

    Andrés-León E, Gómez-López G, Pisano DG. Prediction of miRNA–mRNA interactions using miRGate. Methods Mol Biol. 2017;1580:225–37.

    Article  Google Scholar 

  48. 48.

    Fabian MR, Sonenberg N, Filipowicz W. Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem. 2010;79:351–79.

    CAS  Article  Google Scholar 

  49. 49.

    Lesch BJ, Page DC. Genetics of germ cell development. Nat Rev Genet. 2012;13:781–94.

    CAS  Article  Google Scholar 

  50. 50.

    Uitterlinden A. An introduction to genome-wide association studies: GWAS for dummies. Semin Reprod Med. 2016;34:196–204.

    CAS  Article  Google Scholar 

  51. 51.

    Fritah S, Niclou SP, Azuaje F. Databases for lncRNAs: a comparative evaluation of emerging tools. RNA. 2014;20:1655–65.

    CAS  Article  Google Scholar 

  52. 52.

    Pang KC, Frith MC, Mattick JS. Rapid evolution of noncoding RNAs: lack of conservation does not mean lack of function. Trends Genet. 2006;22:1–5.

    CAS  Article  Google Scholar 

  53. 53.

    Liang M, Hu K, He C, Zhou J, Liao Y. Upregulated lncRNA Gm2044 inhibits male germ cell development by acting as miR-202 host gene. Anim Cells Syst. 2019;23:128–34.

    CAS  Article  Google Scholar 

  54. 54.

    Wang J, Chen J, Sen S. MicroRNA as biomarkers and diagnostics. J Cell Physiol. 2015;231:25–30.

    Article  Google Scholar 

  55. 55.

    Qin Y, Ji J, Du G, Wu W, Dai J, Hu Z, et al. Comprehensive pathway-based analysis identifies associations of BCL2, GNAO1 and CHD2 with non-obstructive azoospermia risk. Hum Reprod. 2014;29:860–6.

    CAS  Article  Google Scholar 

  56. 56.

    Cheng P, Chen H, Zhang RP, Liu S, Zhou-Cun A. Polymorphism in DNMT1 may modify the susceptibility to oligospermia. Reprod BioMed Online. 2014;28:644–9.

    CAS  Article  Google Scholar 

  57. 57.

    Abid S, Gokral J, Maitra A, Meherji P, Kadam S, Pires E, et al. Altered expression of progesterone receptors in testis of infertile men. Reprod BioMed Online. 2008;17:175–84.

  58. 58.

    Sen S, Dixit A, Thakur C, Gokral J, Hinduja I, Zaveri K, et al. Association of progesterone receptor gene polymorphism with male infertility and clinical outcome of ICSI. J Assist Reprod Genet. 2013;30:1133–9.

  59. 59.

    Puga Molina LC, Pinto NA, Torres Rodríguez P, Romarowski A, Vicens Sanchez A, Visconti PE, et al. Essential role of CFTR in PKA-dependent phosphorylation, alkalinization, and hyperpolarization during human sperm capacitation. J Cell Physiol. 2017;232:1404–14.

  60. 60.

    Tamburino L, Guglielmino A, Venti E, Chamayou S. Molecular analysis of mutations and polymorphisms in the CFTR gene in male infertility. Reprod BioM Online. 2008;17:27–35.

    CAS  Article  Google Scholar 

  61. 61.

    Lachance C, Goupil S, Leclerc P. Stattic V, a STAT3 inhibitor, affects human spermatozoa through regulation of mitochondrial activity. J Cell Physiol. 2012;228:704–13.

    Article  Google Scholar 

Download references


This work is supported by the Spermogene project which is co-financed by the European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship, and Innovation, under the call RESEARCH–CREATE–INNOVATE (Grant number Τ1ΕΔΚ-02787).

Author information




Maria-Anna Kyrgiafini was responsible for all data analysis and composing the manuscript along with participating in reviewing important details. Maria Markantoni participated in the creation of the study concept and in data interpretation. Theologia Sarafidou was responsible for article significant revisions. Alexia Chatziparasidou and Nicolas Christoforidis were responsible for all samples acquisition along with consent forms. Zissis Mamuris was responsible for the study concept and funding acquisition, along with article revisions.

Corresponding author

Correspondence to Zissis Mamuris.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethics approval

All the participants were informed about the study and they gave their consent in order to participate by filling out a questionnaire along with the consent form. Both the study and the consent procedure were approved by the ethics committee of the Medical Faculty of the University of Thessaly.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Maria-Anna Kyrgiafini and Maria Markantoni are joint first authors

Electronic supplementary material


(DOCX 1199 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kyrgiafini, MA., Markantoni, M., Sarafidou, T. et al. Genome-wide association study identifies candidate markers related to lincRNAs associated with male infertility in the Greek population. J Assist Reprod Genet 37, 2869–2881 (2020).

Download citation


  • GWAS
  • Male infertility
  • Greek population
  • lincRNA