P–056 To graft or not to graft? Intratesticular grafting of testicular tissue from Klinefelter boys to the mouse testis as possible novel in vivo model
Authors: Willems M , Sesenhausen P , Gies I , Vloeberghs V , Schepper J D , Tournaye H , Goosens E , Va. Saen D 
Published: August 2021

Abstract

Study question
Can intratesticular transplanted testis tissue from Klinefelter boys to the mouse testis be used to study the mechanisms behind testicular fibrosis?

Summary answer
Grafting of testicular tissue from Klinefelter boys to the mouse testis is not a valuable new in vivo model to study Klinefelter-related testicular fibrosis.

What is known already
Klinefelter syndrome (KS; 47, XXY) affects 1–2 in 1000 males. Most KS men suffer from azoospermia due to a loss of spermatogonial stem cells. Additionally, testicular fibrosis is detected from puberty onwards. However, mechanisms responsible for fibrosis and germ cell loss remain unknown. An optimal in vivo model to study the KS testicular fibrotic process is not available.
This study aimed to evaluate a possible in vivo model to study KS-related testicular fibrosis. In addition, the effect of the mast cell blocker ketotifen, which showed positive effects on fertility in infertile non-KS patients, was evaluated in this graft model.

Study design, size, duration
First, the survival time of the KS graft was established, since it was the first time KS tissue was transplanted to the mouse testis. Testes were collected after two, four, six and eight weeks after which histological and immunohistochemical evaluations were performed. Next, the effect of daily ketotifen injections on the fibrotic appearance of intratesticular grafted testicular tissue from KS and controls was evaluated.

Participants/materials, setting, methods
Testicular biopsy samples from pre- and peripubertal KS (n = 22) and age-matched control samples (n = 22) were transplanted to the testes of six weeks old Swiss Nu/Nu mice (n = 22). Prior to grafting, testicular tissue pieces were cultured in vascular endothelial growth factor (VEGF) for five days. Next, tissues were transplanted to the mouse testes. Testicular transplants were analysed by immunohistochemistry. In the second experiment, mice were given daily subcutaneous injections of ketotifen or saline.

Main results and the role of chance
Four weeks after transplantation, all KS grafts could still be retrieved. At a later timepoint, degeneration of the tissue could be detected. In the grafts, recovered four weeks after transplantation, about 30% of the tubules in peripubertal grafts showed a good integrity, while in the prepubertal tissue, 83% of the tubules were intact. A fibrotic score was assigned to each graft. No significant changes in fibrotic score was observed between testicular biopsies before or after transplantation. However, an increased (p &lt; 0.01) fibrotic score was observed after in-vitro treatment with VEGF both in control and KS tissue. Based on recovery and tubule integrity grafts were recovered after four weeks in the second experiment. Treatment with ketotifen did not result in significant histological differences compared to non-treated grafts (KS and control tissue).
The survival potential of grafts from KS testicular biopsies of pre- and peripubertal boys was patient- and age-dependent. After four weeks, most KS tissue starts to degenerate. In prepubertal tissue, seminiferous tubules were mostly intact, while tissue from adolescent boys was impaired. Interestingly, no loss of germ cells was observed after transplantation of the testicular tissue.

Limitations, reasons for caution
The availability of tissue from young KS patients is very scarce, leading to a low number of included patients (n = 8). Testicular tissue pieces from the same patient were included to evaluate the differences before and after transplantation. However, histological variability between testicular tissue biopsy pieces is well-known in KS patients.

Wider implications of the findings
Since testicular tissue from KS boys, transplanted to the mouse testes, already starts to degenerate after four weeks and the integrity is not optimal, we conclude that this is not a valuable model for future studies. In vitro models to study the KS-testicular fibrosis should be investigated.

Trial registration number
NA