Evaluation of immune status in testis and macrophage polarization associated with testicular damage in patients with nonobstructive azoospermia

Am J Reprod Immunol. 2021 Nov;86(5):e13481. doi: 10.1111/aji.13481. Epub 2021 Aug 9.


Objective: Immune cells residing in the testicular interstitial space form the immunological microenvironment of the testis. They are assumed to play a role in maintaining testicular homeostasis and immune privilege. However, the immune status and related cell polarization in patients with nonobstructive azoospermia (NOA) remains poorly characterized. System evaluation of the testis immunological microenvironment in NOA patients may help to reveal the mechanisms of idiopathic azoospermia.

Study design: The gene expression patterns of immune cells in normal human testes were systematically analyzed by single-cell RNA sequencing (scRNA-seq) and preliminarily verification by the human protein atlas (HPA) online database. The immune cell infiltration profiles and immune status of patients with NOA was analyzed by single-sample gene set enrichment analysis (ssGSEA) and gene set variation analysis (GSVA) based on four independent public microarray datasets (GSE45885, GSE45887, GSE9210, and GSE145467), obtained from Gene Expression Omnibus (GEO) online database. The relationship between immune cells and spermatogenesis score was further analyzed by Spearman correlation analysis. Finally, immunohistochemistry (IHC) staining was performed to identify the main immune cell types and their polarization status in patients with NOA.

Results: Both scRNA-seq and HPA analysis showed that testicular macrophages represent the largest pool of immune cells in the normal testis, and also exhibit an attenuated inflammatory response by expressing high levels of tolerance proteins (CD163, IL-10, TGF-β, and VEGF) and reduced expression of TLR signaling pathway-related genes. Correlation analysis revealed that the testicular immune score and macrophages including M1 and M2 macrophages were significantly negatively correlated with spermatogenesis score in patients with NOA (GSE45885 and GSE45887). In addition, the number of M1 and M2 macrophages was significantly higher in patients with NOA (GSE9210 and GSE145467) than in normal testis. GSVA analysis indicated that the immunological microenvironment in NOA tissues was manifested by activated immune system and pro-inflammatory status. IHC staining results showed that the number of M1 and M2 macrophages was significantly higher in NOA tissues than in normal testis and negatively correlated with the Johnson score.

Conclusion: Testicular macrophage polarization may play a vital role in NOA development and is a promising potential therapeutic target.

Keywords: M1 macrophages; M2 macrophages; immunological microenvironment; nonobstructive azoospermia; testicular macrophages.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azoospermia / genetics
  • Azoospermia / immunology*
  • Azoospermia / metabolism
  • Azoospermia / pathology
  • Databases, Genetic
  • Gene Expression Profiling
  • Humans
  • Macrophages / immunology*
  • Macrophages / metabolism
  • Male
  • Phenotype
  • RNA-Seq
  • Signal Transduction
  • Single-Cell Analysis
  • Spermatogenesis*
  • Testis / immunology*
  • Testis / metabolism
  • Testis / pathology
  • Transcriptome

Supplementary concepts

  • Azoospermia, Nonobstructive