Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia

Hum Reprod. 2024 May 2;39(5):1131-1140. doi: 10.1093/humrep/deae057.


Study question: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype?

Summary answer: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role.

What is known already: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA.

Study design, size, duration: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021.

Participants/materials, setting, methods: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data.

Main results and the role of chance: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group.

Limitations, reasons for caution: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses.

Wider implications of the findings: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA.

Study funding/competing interest(s): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose.

Trial registration number: N/A.

Keywords: MLPA; copy-number variation; mutation; non-obstructive azoospermia; spermatogenic failure; whole exome sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Azoospermia* / genetics
  • Cell Cycle Proteins*
  • DNA Copy Number Variations*
  • Exome Sequencing
  • Humans
  • Japan
  • Karyotyping
  • Male
  • Mutation


  • TEX11 protein, human
  • Cell Cycle Proteins

Supplementary concepts

  • Azoospermia, Nonobstructive