To determine associations between genomic DNA methylation in testicular cells and azoospermia in human males.
This was a case-control study investigating the differences and conservations in DNA methylation, genome-wide DNA methylation, and bulk RNA-Seq for transcriptome profiling using testicular biopsy tissues from NOA and OA patients. Differential methylation and different conserved methylation regions associated with azoospermia were identified by comparing genomic DNA methylation of testicular seminiferous cells derived from NOA and OA patients.
The genome methylation modification of testicular cells from NOA patients was disordered, and the reproductive-related gene expression was significantly different.
Our findings not only provide valuable knowledge of human spermatogenesis but also paved the way for the identification of genes/proteins involved in male germ cell development. The approach presented in this report provides a powerful tool to identify responsible biomolecules, and/or cellular changes (e.g., epigenetic abnormality) that induce male reproductive dysfunction such as OA and NOA.
This is a preview of subscription content, log in to check access.
Buy single article
Instant access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Green CD, Ma Q, Manske GL, Shami AN, Zheng X, Marini S, et al. A comprehensive roadmap of murine spermatogenesis defined by single-cell RNA-Seq. Dev Cell. 2018;46(5):651–67.e10.
Liu Y, Giannopoulou EG, Wen D, Falciatori I, Elemento O, Allis CD, et al. Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells. Nat Commun. 2016;7:11275. https://doi.org/10.1038/ncomms11275.
Wang M, Liu X, Chang G, Chen Y, An G, Yan L, et al. Single-cell RNA sequencing analysis reveals sequential cell fate transition during human spermatogenesis. Cell Stem Cell. 2018;23(4):599–614.e4. https://doi.org/10.1016/j.stem.2018.08.007.
Hermann BP, Cheng K, Singh A, Roa-De La Cruz L, Mutoji KN, Chen IC, et al. The mammalian spermatogenesis single-cell transcriptome, from spermatogonial stem cells to spermatids. Cell Rep. 2018;25(6):1650–67.e8.
Guo J, Grow EJ, Mlcochova H, Maher GJ, Lindskog C, Nie X, et al. The adult human testis transcriptional cell atlas. Cell Res. 2018;28(12):1141–57.
Schlegel PN. Evaluation of male infertility. Minerva Ginecol. 2009;61(4):261–83.
Arafat M, Har-Vardi I, Harlev A, Levitas E, Zeadna A, Abofoul-Azab M, et al. Mutation in TDRD9 causes non-obstructive azoospermia in infertile men. J Med Genet. 2017;54(9):jmedgenet-2017-104514.
Riera-Escamilla A, Enguita-Marruedo A, Moreno-Mendoza D, Chianese C, Sleddens-Linkels E, Contini E, et al. Sequencing of a ‘mouse azoospermia’ gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest. Hum Reprod. 2019;34(6):978–88.
Khazamipour N, Noruzinia M, Fatehmanesh P, Keyhanee M, Pujol P. MTHFR promoter hypermethylation in testicular biopsies of patients with non-obstructive azoospermia: the role of epigenetics in male infertility. Hum Reprod. 2009;24(9):2361–4. https://doi.org/10.1093/humrep/dep194.
Arafat M, Har-Vardi I, Harlev A, Levitas E, Zeadna A, Abofoul-Azab M, et al. Mutation in TDRD9 causes non-obstructive azoospermia in infertile men. J Med Genet. 2017;54(9):633–9. https://doi.org/10.1136/jmedgenet-2017-104514.
Jiang T, Wang Y, Zhu M, Wang Y, Huang M, Jin G, et al. Transcriptome-wide association study revealed two novel genes associated with nonobstructive azoospermia in a Chinese population. Fertil Steril. 2017;108(6):1056–62 e4. https://doi.org/10.1016/j.fertnstert.2017.09.023.
Riera-Escamilla A, Enguita-Marruedo A, Moreno-Mendoza D, Chianese C, Sleddens-Linkels E, Contini E, et al. Sequencing of a ‘mouse azoospermia’ gene panel in azoospermic men: identification of RNF212 and STAG3 mutations as novel genetic causes of meiotic arrest. Hum Reprod. 2019;34(6):978–88. https://doi.org/10.1093/humrep/dez042.
Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16(1):6–21.
Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science. 2001;293(5532):1068–70.
Donkin I, Versteyhe S, Ingerslev LR, Qian K, Mechta M, Nordkap L, et al. Obesity and bariatric surgery drive epigenetic variation of spermatozoa in humans. Cell Metab. 2016;23(2):369–78.
Wei Y, Schatten H, Sun QY. Environmental epigenetic inheritance through gametes and implications for human reproduction. Hum Reprod Update. 2015;21(2):194–208.
Sujit KM, Sarkar S, Singh V, Pandey R, Agrawal NK, Trivedi S, et al. Genome-wide differential methylation analyses identifies methylation signatures of male infertility. Hum Reprod. 2018;33(12):2256–67. https://doi.org/10.1093/humrep/dey319.
Cheng YS, Wee SK, Lin TY, Lin YM. MAEL promoter hypermethylation is associated with de-repression of LINE-1 in human hypospermatogenesis. Hum Reprod. 2017;32(12):2373–81. https://doi.org/10.1093/humrep/dex329.
Ramasamy R, Ridgeway A, Lipshultz LI, Lamb DJ. Integrative DNA methylation and gene expression analysis identifies discoidin domain receptor 1 association with idiopathic nonobstructive azoospermia. Fertil Steril. 2014;102(4):968–73 e3. https://doi.org/10.1016/j.fertnstert.2014.06.028.
Zhu Z, Chong L, Yang S, Ruhui T, Wang J, Qingqing Y, et al. Dynamics of the transcriptome during human spermatogenesis: predicting the potential key genes regulating male gametes generation. Sci Rep. 2016;6:19069.
Tingting J, Wang Y, Zhu M, Wang Y, Mingtao H, Guangfu J, et al. Transcriptome-wide association study revealed two novel genes associated with nonobstructive azoospermia in a Chinese population. Fertil Steril. 2017;108(6):1056–62. e4.
Li Z, Zhuang X, Zeng J, Tzeng CM. Integrated analysis of DNA methylation and mRNA expression profiles to identify key genes in severe oligozoospermia. Front Physiol. 2017;8:261. https://doi.org/10.3389/fphys.2017.00261.
McLachlan RI, Rajpert-De Meyts E, Hoei-Hansen CE, de Kretser DM, Skakkebaek NE. Histological evaluation of the human testis--approaches to optimizing the clinical value of the assessment: mini review. Hum Reprod. 2007;22(1):2–16. https://doi.org/10.1093/humrep/del279.
Qian G, Bao Y, Song D, Chen N, Lin Z. SOHLHs might be gametogenesis-specific bHLH transcriptional regulation factors in Crassostrea gigas. Front Physiol. 2019;10:594. https://doi.org/10.3389/fphys.2019.00594.
Lobaccaro J-MA, Han F, Dong Y, Liu W, Ma X, Shi R, et al. Epigenetic regulation of Sox30 is associated with testis development in mice. PLoS One. 2014;9(5):e97203. https://doi.org/10.1371/journal.pone.0097203.
Khezri A, Narud B, Stenseth E-B, Johannisson A, Myromslien FD, Gaustad AH, et al. DNA methylation patterns vary in boar sperm cells with different levels of DNA fragmentation. BMC Genomics. 2019;20(1):897. https://doi.org/10.1186/s12864-019-6307-8.
Huang WY, Hsu SD, Huang HY, Sun YM, Chou CH, Weng SL, et al. MethHC: a database of DNA methylation and gene expression in human cancer. Nucleic Acids Res. 2015;43(Database issue):D856–61. https://doi.org/10.1093/nar/gku1151.
Jones PA. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 2012;13(7):484–92. https://doi.org/10.1038/nrg3230.
Yang X, Han H, De Carvalho DD, Lay Fides D, Jones Peter A, Liang G. Gene body methylation can alter gene expression and is a therapeutic target in cancer. Cancer Cell. 2014;26(4):577–90. https://doi.org/10.1016/j.ccr.2014.07.028.
Zhang C, Xue P, Gao L, Chen X, Lin K, Yang X, et al. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility. FASEB J. 2016;30(10):3424–40. https://doi.org/10.1096/fj.201500167R.
Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature. 2009;462(7270):222–5. https://doi.org/10.1038/nature08562.
Tung JY, Rosen MP, Nelson LM, Turek PJ, Witte JS, Cramer DW, et al. Variants in Deleted in AZoospermia-Like (DAZL) are correlated with reproductive parameters in men and women. Hum Genet. 2006;118(6):730–40. https://doi.org/10.1007/s00439-005-0098-5.
Navarro-Costa P, Nogueira P, Carvalho M, Leal F, Cordeiro I, Calhaz-Jorge C, et al. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm. Hum Reprod. 2010;25(10):2647–54. https://doi.org/10.1093/humrep/deq200.
Luangpraseuth-Prosper A, Lesueur E, Jouneau L, Pailhoux E, Cotinot C, Mandon-Pepin B. TOPAZ1, a germ cell specific factor, is essential for male meiotic progression. Dev Biol. 2015;406(2):158–71. https://doi.org/10.1016/j.ydbio.2015.09.002.
Li Q, Li Y, Yang S, Huang S, Yan M, Ding Y, et al. CRISPR-Cas9-mediated base-editing screening in mice identifies DND1 amino acids that are critical for primordial germ cell development. Nat Cell Biol. 2018;20(11):1315–25. https://doi.org/10.1038/s41556-018-0202-4.
Zangen D, Kaufman Y, Banne E, Weinberg-Shukron A, Abulibdeh A, Garfinkel BP, et al. Testicular differentiation factor SF-1 is required for human spleen development. J Clin Invest. 2014;124(5):2071–5. https://doi.org/10.1172/JCI73186.
Lourenco D, Brauner R, Lin L, De Perdigo A, Weryha G, Muresan M, et al. Mutations in NR5A1 associated with ovarian insufficiency. N Engl J Med. 2009;360(12):1200–10. https://doi.org/10.1056/NEJMoa0806228.
Deqiang D, Jiali L, Kunzhe D, Uros M, Hess Rex A, Huirong X, et al. PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice. Nat Commun. 2017;8(1):819.
Levy A, Schwartz S, Ast G. Large-scale discovery of insertion hotspots and preferential integration sites of human transposed elements. Nucleic Acids Res. 2010;38(5):1515–30.
Rigal M, Mathieu O. A “mille-feuille” of silencing: epigenetic control of transposable elements. Biochim Biophys Acta. 2011;1809(8):452–8.
Kee K, Angeles VT, Flores M, Nguyen HN, Reijo Pera RA. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature. 2009;462(7270):222–5.
Navarro-Costa P, Nogueira P, Carvalho M, Leal F, Cordeiro I, Calhaz-Jorge C, et al. Incorrect DNA methylation of the DAZL promoter CpG island associates with defective human sperm. Human Reprod (Oxford, England). 2010;25(10):2647–54.
Kato Y, Katsuki T, Kokubo H, Masuda A, Saga Y. Dazl is a target RNA suppressed by mammalian NANOS2 in sexually differentiating male germ cells. Nat Commun. 2016;7(undefined):11272.
Zhang C, Xue P, Gao L, Chen X, Lin K, Yang X, et al. Highly conserved epigenetic regulation of BOULE and DAZL is associated with human fertility. FASEB J : official publication of the Federation of American Societies for Experimental Biology. 2016;30(10):3424–40.
Li H, Liang Z, Yang J, Wang D, Wang H, Zhu M, et al. DAZL is a master translational regulator of murine spermatogenesis. Natl Sci Rev. 2019;6(3):455–68.
Zhou L, Canagarajah B, Zhao Y, Baibakov B, Tokuhiro K, Maric D, et al. BTBD18 regulates a subset of piRNA-generating loci through transcription elongation in mice. Dev Cell. 2017;40(5):453–66.e5.
Yamaji M, Jishage M, Meyer C, Suryawanshi H, Der E, Yamaji M, et al. DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Nature. 2017;543(7646):568–72.
Li H, Wu C, Gu X, Xiong C. A novel application of cell-free seminal mRNA: non-invasive identification of the presence of germ cells or complete obstruction in men with azoospermia. Human Reprod (Oxford, England). 2012;27(4):991–7.
Luangpraseuth-Prosper A, Lesueur E, Jouneau L, Pailhoux E, Cotinot C, Mandon-Pépin B. TOPAZ1, a germ cell specific factor, is essential for male meiotic progression. Dev Biol. 2015;406(2):158–71.
Lourenço D, Brauner R, Lin L, De Perdigo A, Weryha G, Muresan M, et al. Mutations in NR5A1 associated with ovarian insufficiency. N Engl J Med. 2009;360(12):1200–10.
Zangen D, Kaufman Y, Banne E, Weinberg-Shukron A, Abulibdeh A, Garfinkel BP, et al. Testicular differentiation factor SF-1 is required for human spleen development. J Clin Invest. 2014;124(5):2071–5.
Gu A, Ji G, Shi X, Long Y, Xia Y, Song L, et al. Genetic variants in Piwi-interacting RNA pathway genes confer susceptibility to spermatogenic failure in a Chinese population. Human Reprod (Oxford, England). 2010;25(12):2955–61.
Gomes Fernandes M, He N, Wang F, Van Iperen L, Eguizabal C, Matorras R, et al. Human-specific subcellular compartmentalization of P-element induced wimpy testis-like (PIWIL) granules during germ cell development and spermatogenesis, Human Reproduction (Oxford, England). 2018;33(2):258–69.
Suzuki A, Niimi Y, Shinmyozu K, Zhou Z, Kiso M, Saga Y. Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development. EMBO Rep. 2016;17(1):37–46.
Kedde M, Strasser MJ, Boldajipour B, Oude Vrielink JA, Slanchev K, le Sage C, et al. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell. 2007;131(7):1273–86.
Feschotte C, Pritham EJ. DNA transposons and the evolution of eukaryotic genomes. Annu Rev Genet. 2007;41(undefined):331–68.
Bennett EA, Keller H, Mills RE, Schmidt S, Moran JV, Weichenrieder O, et al. Active Alu retrotransposons in the human genome. Genome Res. 2008;18(12):1875–83.
S.F and C.Y.C conceived and designed the study. C.X and J.F collected the sample. XL.W and CH.L performed the experiments. LF.H and YM.C analyzed and interpreted the data. XL.W wrote the manuscript and S.F and C.Y.C revised the manuscript. All authors discussed the results and implications of the study.
This work was supported by The National Key Research and Development Program of China (2018YFC1003500 to F.S), The National Natural Science Foundation of China (81671510 to F.S), and The Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX19_2027).
The experiments using human materials were approved by Nantong University. Informed consent was obtained from all human subjects.
Conflict of interest
The authors declare that they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Wu, X., Luo, C., Hu, L. et al. Unraveling epigenomic abnormality in azoospermic human males by WGBS, RNA-Seq, and transcriptome profiling analyses. J Assist Reprod Genet (2020). https://doi.org/10.1007/s10815-020-01716-7
- Non-obstructive azoospermia (NOA)
- Obstructive azoospermia (OA)
- DNA methylation
- Bulk transcriptome