Examining potential mechanisms of testicular fibrosis in Klinefelter Syndrome: A review of current understanding

Bradshaw, Aaron; Deebel, Nicholas A.; Xu, Mark C.; Kogan, Stanley J.; Atala, Anthony; Sadri‐Ardekani, Hooman

doi:10.1111/andr.13327

Cited by 4 publications

(1 citation statement)

References 29 publications

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“…In addition to changes in the vasculature and the blood–testis barrier (BTB), previous studies of KS testes have identified an increased number of immune cells and increased inflammation as the possible foundation for the observed testicular fibrosis ( D'Aurora et al , 2015 ; Willems et al , 2020 ; Bradshaw et al , 2023 ). Cell-specific changes in gene expression pointed towards an increased signaling interaction strength of both macrophages and MCs in KS and NOA compared to CTRLs, and a marked increase in the incoming lymphocyte interaction strength in KS.…”

Section: Discussionmentioning

confidence: 99%

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

Johannsen,

Skakkebæk,

Kalucka

et al. 2023

Human Reproduction

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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.

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Section: Discussionmentioning

confidence: 99%

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

Johannsen,

Skakkebæk,

Kalucka

et al. 2023

Human Reproduction

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Long-term culture of human Sertoli cells from adult Klinefelter patients as a first step to develop new tools for unravelling the testicular physiopathology

Giudice,

Kanbar,

Poels

et al. 2024

Human Reproduction

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STUDY QUESTION Are Sertoli cells (SCs) from adult Klinefelter men (47,XXY) capable of proliferating in vitro and maintaining their main phenotypical and functional characteristics as do SCs from adult 46,XY patients? SUMMARY ANSWER Isolated SCs from patients with Klinefelter syndrome (KS) can be expanded in vitro while maintaining their characteristics and a stable karyotype, similar to SCs from 46,XY patients. WHAT IS KNOWN ALREADY The mechanism leading to testicular tissue degeneration in KS is still unknown. A few recent studies highlight the main role played by SCs in the physiopathology of the disease, but new study models based on co-culture or testicular organoids are needed to further understand the SC’s involvement in the mechanism of testicular degeneration and fibrosis, and to find therapeutical targets. KS SC expansion could be the first step towards developing such in vitro study models. SCs have been isolated from 46,XY men and expanded in vitro while maintaining the expression of phenotypical and functional markers, but propagation of SCs from KS men has not been achieved yet. STUDY DESIGN, SIZE, DURATION Testicular tissue was obtained during a testicular sperm extraction procedure for infertility treatment between 2019 and 2021 from three azoospermic adult KS (47,XXY) men (33±3.6 years old) and from three control patients (46,XY) (36±2 years old) presenting with obstructive azoospermia. SCs isolated from frozen-thawed tissue of KS and 46,XY patients were cultured for 60 days and compared. All patients signed an informed consent according to the ethical board approval of the study protocol. PARTICIPANTS/MATERIALS, SETTING, METHODS Testicular biopsies obtained from KS (n = 3) and 46,XY (n = 3) adult patients were slow-frozen. After tissue thawing SCs were isolated using a double-step enzymatic digestion and differential plating, and cultured for 60 days in DMEM medium containing FBS. Analyses were performed at different culture times (passages 5 (P5) and 10 (P10)). Quantification of cells using immunofluorescence (IF) for cell type-specific markers (Sox9, GATA4, ACTA2, INSL3, MAGEA4), SCs characterization using both IF and quantitative real-time PCR for GDNF, BMP4, AR and CLDN11 and cells karyotyping were performed. MAIN RESULTS AND THE ROLE OF CHANCE We demonstrate for the first time that a small population of human SCs isolated from frozen-thawed testis of adult KS patients can be expanded in vitro while retaining expression of characteristic markers of SCs and the 47,XXY karyotype, and exhibiting cell-specific functional proteins and gene expression (GDNF, BMP4, AR, and CLDN11) after 60 days in culture. At P10, 83.39 ± 4.2% of cultured cells from KS men and 85.34 ± 4.1% from 46,XY men expressed Sox9, and 88.8 ± 3.9% of KS cells versus 82.9 ± 3.2% of the control cells were positive for GATA4 without any differences between two groups; both Sox9 and GATA4 are typical SC markers. No differences were found between KS and 46,XY SCs in vitro in terms of cells expansion (exponential growth between P1 and P10 with an average cell count of 2.8±1.5×107 versus 3.8±1.2×107 respectively for the KS and control groups at P10). There was no significant statistical difference for functional proteins and genes expressions (GDNF, BMP4, AR, and CLDN11) neither between KS SCs and control SCs nor between P5 and P10. LIMITATIONS, REASONS FOR CAUTION The small number of donor samples is a limitation but it is due to limited availability of tissue for research in KS populations. Although no differences were observed in SCs function in the culture of isolated SCs after 60 days, the possibility of a SCs dysfunction needs to be investigated in more complex 3-dimensional models allowing the establishment of a proper cell organization and further analyses of cell functions and interactions during longer culture periods. WIDER IMPLICATIONS OF THE FINDINGS The demonstration of the possibility to propagate KS SCs in vitro could be useful to build new in vitro models for deciphering testicular cell interactions, determining deficient signalling pathways involved in impaired spermatogenesis, and identifying targets for infertility treatment in KS. As the cell numbers achieved in this study are higher than cell numbers used to develop testicular organoids, we may expect to be able to understand the behaviour and physiopathology of SCs in the disease during the long-term culture of these organoids. Such models could be further applied to understand other causes of deficiencies in seminiferous tubules. STUDY FUNDING/COMPETING INTEREST(S) M.G.G is funded by a grant from the Cliniques Universitaires Saint-Luc (FRC) for the research project on Klinefelter Syndrome Physiopathology. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER NCT05997706.

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Testicular fibrosis pathology, diagnosis, pathogenesis, and treatment: A perspective on related diseases

Xu,

Hu,

Chen

et al. 2024

Andrology

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Testicular fibrosis is a chronic and progressive condition characterized by the excessive deposition of extracellular matrix proteins. This process leads to fibrotic remodeling, damage to testicular tissue, and the irreversible loss of male reproductive function. However, there is currently a lack of comprehensive reviews systematically elucidating the pathology, diagnosis, pathogenesis, and treatment of testicular fibrosis from the perspectives of different related diseases. This review addresses these aspects of testicular fibrosis, with a particular emphasis on elucidating the underlying mechanisms of testicular cells. It provides insights that can be relevant for future research and clinical interventions.

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Examining potential mechanisms of testicular fibrosis in Klinefelter Syndrome: A review of current understanding

Cited by 4 publications

References 29 publications

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

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

Long-term culture of human Sertoli cells from adult Klinefelter patients as a first step to develop new tools for unravelling the testicular physiopathology

Testicular fibrosis pathology, diagnosis, pathogenesis, and treatment: A perspective on related diseases

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