The testicular microvasculature in Klinefelter syndrome is immature with compromised integrity and characterized by excessive inflammatory cross-talk.

Study Question: Does Klinefelter syndrome (KS) lead to a distinct gene expression pattern at single-cell level in the testes that could provide insight into the reported microvascular dysfunction in the testes?

Summary Answer: A distinct gene expression pattern within microvascular-associated cells of males with KS suggests excessive endothelial cell (EC) activation, disorganized vessel formation, and the presence of immature vessels with compromised integrity.

What Is Known Already: Recent studies show that males with KS exhibit microvascular dysfunction in their testes, which affects blood flow and is associated with lower circulating levels of testosterone.

Study Design, Size, Duration: A comparative cross-sectional study of males with KS (n = 6), non-obstructive azoospermia (NOA) (n = 5), cryptozoospermia (n = 3), and controls (n = 15) was carried out.

Participants/materials, Setting, Methods: We analyzed publicly available single-cell RNA sequencing data of testicular cells from males with KS, males with NOA, males with cryptozoospermia, and controls. The integration of these datasets allowed us to analyze gene expression profiles and communication patterns among the cell types within the testis and to identify capillary ECs to investigate changes at the microvascular level.

Main Results And The Role Of Chance: Rooted in changes at the single-cell level, our study demonstrates a shift in gene expression forming the foundation for altered cellular communication, microvascular remodeling, and pro-inflammatory responses within the testes of males with KS. We identified genes that were dysregulated in capillary ECs from males with KS (Padj < 0.05). Specifically, the unique microvascular gene expression in males with KS indicated enhanced capillary EC activation and increased inflammatory cross-talk, leading to impaired vessel maturation and increased EC barrier permeability.

Limitations, Reasons For Caution: Our study is constrained by an unbalanced design, with varying sample sizes and number of cells within each group. We acknowledge the restricted access to clinical information. In addition, our findings were deduced from changes in gene expression, which limits us to infer potential biological consequences arising from these alterations. Furthermore, the absence of a pre-pubertal age group limits the generalizability of our findings and warrants further investigation.

Wider Implications Of The Findings: This study offers novel insights into the testicular pathophysiology in KS and underscores the potential contribution of microvascular dysfunction to the hypogonadism and infertility observed in males with KS. While this study aims to better understand the microvascular dysfunction in KS, the precise connections to testosterone deficiency and testicular atrophy remain to be fully elucidated.

Study Funding/competing Interest(s): A.S. was supported by the Independent Research Fund Denmark (0134-00130B). C.H.G. was supported by Novo Nordisk Foundation (NNF15OC0016474, NNF20OC0060610), 'Fonden til lægevidenskabens fremme', the Familien Hede Nielsen foundation and the Independent Research Fund Denmark (0134-00406A). E.B.J. was supported by Aarhus University and E.B.J. and C.H.G by the Independent Research Fund Denmark (2096-00165A). J.M.K. was supported by Lundbeckfonden (R307-2018-3667), Carlsberg Fonden (CF19-0687), Novo Nordisk Fonden (0073440) and Steno Diabetes Center Aarhus (SDCA). The authors declare no conflicts of interest.

Trial Registration Number: N/A.