Intratesticular xenografting of Klinefelter pre-pubertal testis tissue as potential model to study testicular fibrosis.

RESEARCH QUESTION: Is intratesticular xenotransplantation a potential ex-vivo model for studying testicular fibrosis related to Klinefelter syndrome? STUDY DESIGN: First, a feasibility study of an ex-vivo model to study testicular fibrosis in patients with Klinefelter syndrome was performed. Testis tissue from boys with pre-pubertal Klinefelter syndrome (n = 3) and controls (n = 2) (<18 years) was grafted to the mouse testis (n = 12) and recovered after 2, 4, 6 and 8 weeks. Part two of this study consisted of a validation of this model, evaluating the effects of the mast cell blocker ketotifen on the histology of the grafts of Klinefelter syndrome (n = 5) and controls (n = 3), transplanted to mice (n = 10), after 4 weeks of ketotifen or saline treatment. Immunohistochemistry determined the histology of the grafts and the presence of mast cells and spermatogonia. RESULTS: The feasibility study showed that 4 weeks after transplantation, all Klinefelter syndrome grafts could be recovered. Later, degeneration was observed. Most recovered grafts showed an intact histology, with 67 ± 12% intact tubules for the Klinefelter syndrome grafts and 65 ± 15% of intact tubules for the control grafts. In the few remaining Klinefelter syndrome grafts, treatment with ketotifen improved testicular histology compared with non-treated grafts. Graft survival was patient dependent. No germ cell loss was observed after transplantation. CONCLUSION: Xenografting could become a model for the longitudinal study of the fibrotic process related to Klinefelter syndrome; however, the current model has a limited survival period and patient-specific differences in histology.

Characterisation of testicular function and spermatogenesis in transgender women.

STUDY QUESTION: Does gender-affirming treatment prevent full spermatogenesis in transgender women (TW)? SUMMARY ANSWER: Adequate hormonal therapy (HT) leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. WHAT IS KNOWN ALREADY: Gender-affirming treatment in transgender individuals may involve gender-affirming HT. The effects on spermatogenesis in TW remain unclear. In order to add information from a referral centre for transgender care, we wish to compare results of earlier studies with our population of TW who received a standard hormone treatment. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study part of the European Network for the Investigation of Gender Incongruence (ENIGI), conducted between 15 February 2010 and 30 September 2015. There were 162 TW were included in the ENIGI study at the Ghent University Hospital in Belgium. Participants are included in ENIGI when they first start HT, and follow-up visits occur over the next 3 years. PARTICIPANTS/MATERIALS, SETTING METHODS: The study included 97 TW who initiated HT with cyproterone acetate (CPA) plus oestrogens and proceeded with gonadectomy at the Ghent University Hospital. Testicular tissue retrieved during gonadectomy was processed and stained for four different germ cell markers by the Biology of the Testis lab at the Vrije Universiteit Brussel. Subsequent immunohistochemical staining was performed for melanoma-associated antigen A4 (MAGE-A4, marker for spermatogonia and early spermatocytes), boule homologue, RNA-binding protein (BOLL, marker for secondary spermatocytes and round spermatids), cAMP-responsive element modulator (CREM, marker for round spermatids) and acrosin (marker for acrosome visualization). Serum levels of sex steroids were measured prior to surgery. MAIN RESULTS AND THE ROLE OF CHANCE: Suppressed testosterone levels (<50 ng/dl) were found in 92% of the participants prior to surgery. The mean time between initiation of HT and surgery was 685 days. In 88% (85/97) of the sections, MAGE-A4 staining was positive. Further staining could not reveal complete spermatogenesis in any participant. LIMITATIONS, REASONS FOR CAUTION: Testicular function of the participants prior to initiation of HT was not assessed, although all participants presented with cisgender male serum testosterone values before initiation of HT. The current study only reports on people using CPA at a fixed dose and may therefore not be applicable to all TW. WIDER IMPLICATIONS OF THE FINDINGS: HT leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. Serum testosterone levels are associated with the sperm maturation rate. It is important to discuss sperm preservation before the start of hormone therapy. If serum testosterone levels remain higher, spermatogenesis may still occur. STUDY FUNDING/COMPETING INTEREST(S): D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2819N). Processing of the testis specimens was funded by the Biology of The Testes (BITE) research group (Department of Reproduction, Genetics and Regenerative medicine at Vrije Universiteit Brussel (VUB)). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Germ cell loss in Klinefelter syndrome: When and why?

Klinefelter syndrome (KS) is a quite common disorder with an incidence of 1-2 in 1,000 new-born males. Most patients are diagnosed in the light of a clinical checkup when consulting a fertility clinic with an unfulfilled child wish. Infertility in KS patients is caused by a massive germ cell loss, leading to azoospermia in more than 90% of the adult patients. Most seminiferous tubules in the adult KS testis are degenerated or hyalinized and testicular fibrosis can be observed, starting from puberty. However, focal spermatogenesis can be found in the testis of some patients. This offers the opportunity to extract spermatozoa from the testis by testicular sperm extraction (TESE). Nevertheless, TESE is only successful in about half of the KS adults seeking to father children. The reason for the germ cell loss remains unclear. To date, it is still debated whether the testicular tissue changes and the germ cell loss seen in KS is directly caused by an altered X-linked gene expression, the altered somatic environment, or a deficiency in the germ cells. In this review, we provide an overview of the current knowledge about the germ cell loss in KS patients.

Testicular immune cells and vasculature in Klinefelter syndrome from childhood up to adulthood.

STUDY QUESTION: Is the distribution of immune cells and the testicular vasculature altered in testicular biopsies from patients with Klinefelter syndrome (KS)? SUMMARY ANSWER: Increased numbers of macrophages and mast cells, an increased expression of decorin and an increased blood vessel density were found in KS samples compared to controls. WHAT IS KNOWN ALREADY: Most KS patients are infertile due to an early germ cell loss. From puberty onwards, testicular fibrosis can be detected. How this fibrotic process is initiated remains unknown. STUDY DESIGN, SIZE, DURATION: In this study, the number of macrophages, mast cells and their secretory products were evaluated in KS, Sertoli cell only (SCO) and control patient samples. The association between immune cell numbers and level of fibrosis in KS tissue was examined. In addition, the vascularization within these testicular tissue biopsies was studied. For immunohistochemical evaluation, KS patients at different stages of testicular development were included: prepubertal (aged 4-7 years; n = 4), peripubertal (aged 11-17 years; n = 21) and adult (aged >18 years; n = 37) patients. In addition, testicular tissue biopsies of adult SCO (n = 33) and control samples for the three KS age groups (prepubertal n = 9; peripubertal n = 5; adult n = 25) were analysed. Gene expression analysis was performed on adult testicular tissue from KS (n = 5), SCO (n = 5) and control (n = 5) patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult (>18 years) KS, SCO and control testicular tissue biopsies were obtained during a testicular sperm extraction procedure. KS peripubertal (11-18 years), prepubertal (<11 years) and age-matched control biopsies were obtained from the biobank of the university hospital. Immunohistochemistry was used to determine the tubular structure (H/PAS), the number of spermatogonia (MAGE-A4), macrophages (CD68) and mast cells (tryptase) and the blood vessel density (Von Willebrand factor). In addition, quantitative real-time polymerase chain reaction was used to determine the expression of secretory products of macrophages and mast cells (tryptase, tumour necrosis factor alpha and decorin). MAIN RESULTS AND THE ROLE OF CHANCE: A significant increase in the number of macrophages (P < 0.0001) and mast cells (P = 0.0008) was found in the peritubular compartment of testes of adult KS patients compared to control samples. However, no association between the number of immune cells and the degree of fibrosis was observed. In adult SCO samples, a significant increase was seen for peritubular macrophage (P < 0.0001) and mast cell (P < 0.0001) numbers compared to control samples. In the interstitial compartment, a significant increase in mast cell number was found in adult SCO samples compared to KS (P < 0.0001) and control (P < 0.0001) tissue. A significant difference (P = 0.0431) in decorin expression could be detected in adult KS compared to control patients. Decorin expression was mostly seen in the walls of the seminiferous tubules. When comparing the vascularization between KS patients and age-matched controls, a significant increase (P = 0.0081) in blood vessel density could be observed only in prepubertal KS testicular tissue. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: As controls for this study, testicular tissue biopsies of men who underwent a vasectomy reversal or orchiectomy were used, but these men may not represent fertile controls. In addition, a high variability in immune cell numbers, secretory products expression and number of blood vessels could be observed amongst all patient samples. WIDER IMPLICATIONS OF THE FINDINGS: Increased numbers of macrophages and mast cells have previously been described in non-KS infertile men. Our results show that these increased numbers can also be detected in KS testicular tissue. However, no association between the number of macrophages or mast cells and the degree of fibrosis in KS samples could be detected. Decorin has previously been described in relation to fibrosis, but it has not yet been associated with testicular fibrosis in KS. Our results suggest a role for this proteoglycan in the fibrotic process since an increased expression was observed in adult KS tissue compared to controls. Impaired vascularization in KS men was suggested to be responsible for the KS-related disturbed hormone levels. Our results show a significant difference in blood vessel density, especially for the smallest blood vessels, between prepubertal KS samples and age-matched controls. This is the first study to report differences between KS and control testicular tissue at prepubertal age. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by grants from the Vrije Universiteit Brussel (E.G.) and the scientific Fund Willy Gepts from the UZ Brussel (D.V.S.). D.V.S. is a post-doctoral fellow of the Fonds voor Wetenschappelijk Onderzoek (FWO; 12M2819N). No conflict of interest is declared for this research project.

Effect of recombinant human vascular endothelial growth factor on testis tissue xenotransplants from prepubertal boys: a three-case study.

RESEARCH QUESTION: Does recombinant human vascular endothelial growth factor (VEGF-165) improve the efficiency of human immature testis tissue (ITT) xenotransplantation? DESIGN: ITT fragments from three prepubertal boys were cultured for 5 days with VEGF-165 or without (control) before xenotransplantation into the testes of immunodeficient mice. Xenotransplants were recovered at 4 and 9 months post-transplantation and vascularization, seminiferous tubule integrity, number of spermatogonia and germ cell differentiation were evaluated by histology and immunohistochemistry. RESULTS: Transplants from donor 1 and donor 2 treated with VEGF demonstrated higher vascular surface (P = 0.004) and vessel density (P = 0.011) overall and contained more intact seminiferous tubules (P = 0.039) with time, compared with controls. The number of spermatogonia was increased over time (P < 0.001) irrespective of treatment and donor, whereas, for the VEGF-treated transplants, the increase was even higher over time (P = 0.020). At 9 months, spermatocytes were present in the xenotransplants, irrespective of treatment. No transplants could be recovered from donor 3, who had already received treatment with cyclosporine for aplastic anaemia before biopsy. CONCLUSIONS: In-vitro pre-treatment of human prepubertal testis tissue with VEGF improved transplant vascularization in two out of three cases, resulting in improved seminiferous tubule integrity and spermatogonial survival during xenotransplantation. Although further studies are warranted, we suggest VEGF to be considered as a factor for improving the efficiency of immature testis tissue transplantation in the future.

Exogenous administration of recombinant human FSH does not improve germ cell survival in human prepubertal xenografts.

In a previous study, meiotic activity was observed in human intratesticular xenografts from peripubertal patients. However, full spermatogenesis could not be established. The present study aimed to evaluate whether the administration of recombinant human FSH could improve the spermatogonial survival and the establishment of full spermatogenesis in intratesticular human xenografts. Human testicular tissue was obtained from six boys (aged 2.5-12.5years). The testicular biopsy was fragmented and one fragment of 1.5-3.0mm(3) was transplanted to the testis of immunodeficient nude mice. Transplanted mice were assigned to different experimental groups to enable evaluation of the effects of FSH administration and freezing. The structural integrity of the seminiferous tubules, the spermatogonial survival and the presence of differentiated cells were evaluated by histology and immunohistochemistry. Freezing or administration of FSH did not influence tubule integrity and germ cell survival in human xenografts. Meiotic germ cells were observed in the xenografts. More tubules containing only Sertoli cells were observed in frozen-thawed grafts, and more tubules with meiotic cells were present in fresh grafts. There was no clear influence of FSH treatment on meiotic differentiation. Administration of FSH did not improve the establishment of full spermatogenesis after intratesticular tissue grafting.

Micro RNA in Semen/Urine from Non-Obstructive Azoospermia Patients as Biomarkers to Predict the Presence of Testicular Spermatozoa and Spermatogonia.

About half of testicular sperm extraction (TESE) procedures in men with non-obstructive azoospermia (NOA), including men with Klinefelter syndrome (KS), are unsuccessful. To avoid unnecessary invasive surgery, biomarkers for spermatozoa were studied. In addition, markers for spermatogonia in testis tissue were explored. This study aimed to find biomarkers in the semen and/or urine of NOA patients to predict the presence of spermatogonia in the testis. Differentially expressed miRNAs were identified (1) between samples from patients with and without a positive TESE procedure as well as (2) between TESE-negative patients with and without spermatogonia. A total of thirteen upregulated miRNAs (ten in seminal plasma and three in urine) were found in the TESE-negative/spermatogonia-positive group compared to the TESE-negative/spermatogonia-negative group. These miRNAs could be potential biomarkers for spermatogonia; however, more research is necessary to validate their predictive power.

Transcriptomic differences between fibrotic and non-fibrotic testicular tissue reveal possible key players in Klinefelter syndrome-related testicular fibrosis.

Klinefelter syndrome (KS; 47,XXY) affects 1-2 in 1000 males. Most men with KS suffer from an early germ cell loss and testicular fibrosis from puberty onwards. Mechanisms responsible for these processes remain unknown. Previous genomics studies on testis tissue from men with KS focused on germ cell loss, while a transcriptomic analysis focused on testicular fibrosis has not yet been performed. This study aimed to identify factors involved in the fibrotic remodelling of KS testes by analysing the transcriptome of fibrotic and non-fibrotic testicular tissue. RNA sequencing was performed to compare the genes expressed in testicular samples with (KS and testis atrophy) and without (Sertoli cell-only syndrome and fertile controls) fibrosis (n = 5, each). Additionally, differentially expressed genes (DEGs) between KS and testis atrophy samples were studied to reveal KS-specific fibrotic genes. DEGs were considered significant when p < 0.01 and log2FC > 2. Next, downstream analyses (GO and KEGG) were performed. Lastly, RNA in situ hybridization was performed to validate the results. The first analysis (fibrotic vs non-fibrotic) resulted in 734 significant DEGs (167 up- and 567 down-regulated). Genes involved in the extracellular structure organization (e.g. VCAM1) were found up-regulated. KEGG analysis showed an up-regulation of genes involved in the TGF-ß pathway. The KS vs testis atrophy analysis resulted in 539 significant DEGs (59 up- and 480 down-regulated). Chronic inflammatory response genes were found up-regulated. The overlap of X-linked DEGs from the two analyses revealed three genes: matrix-remodelling associated 5 (MXRA5), doublecortin (DCX) and variable charge X-Linked 3B (VCX3B). RNA in situ hybridization showed an overexpression of VCAM1, MXRA5 and DCX within the fibrotic group compared with the non-fibrotic group. To summarize, this study revealed DEGs between fibrotic and non-fibrotic testis tissue, including VCAM1. In addition, X-linked fibrotic genes were revealed, e.g. MXRA5, DCX and VCX3B. Their potential role in KS-related testicular fibrosis needs further study.

Mouse spermatogonial stem cells obtain morphologic and functional characteristics of hematopoietic cells in vivo.

BACKGROUND: The aim of this study was to explore the plasticity and transdifferentiation potential of murine spermatogonial stem cells (SSCs) into hematopoietic cells. METHODS: GFP(+)CD49f(+)H-2K(b-) SSCs of male donor mice were isolated and injected into the bone marrow (BM) of Busulfan-treated GFP(-) female mice. Twelve weeks post-transplantation, the recipients were sacrificed and their BM, peripheral blood (PB) and spleen (SL) cells were collected and evaluated by phenotypical methods, i.e. fluorescence-activated cell sorting, immunohistochemistry and fluorescence in situ hybridization, and functional assays, i.e. colony-forming units assay and intra-BM transplantation. RESULTS: Green fluorescent protein (GFP)- and Y chromosome-positive cells were observed in the BM, PB and SL of transplanted female mice. These cells presented phenotypical and functional characteristics of hematopoietic cells in vitro and in vivo. CONCLUSIONS: Our results indicate that SSCs have the potential to transdifferentiate into hematopoietic cells in vivo.

Characterization of the stem cell niche components within the seminiferous tubules in testicular biopsies of Klinefelter patients.

OBJECTIVE: To characterize the tubular environment in testicular biopsy tissues from patients with Klinefelter syndrome (KS). DESIGN: Observational immunohistochemical study. SETTING: Academic research unit. PATIENT(S): Males with KS and controls at different developmental time points: fetal, prepubertal, peripubertal, and adult. INTERVENTION(S): Immunohistochemical analysis of testicular biopsies samples to characterize maturation of Sertoli cells and tubular wall components-peritubular myoid cells (PTMC) and extracellular matrix (ECM) proteins. MAIN OUTCOME MEASURE(S): Intensity of antimüllerian hormone staining; proportion of Sertoli cells expressing androgen receptor (AR); and expression of tubular wall markers as characterized by identifying abnormal staining patterns. RESULT(S): Decreased expression for alpha smooth muscle actin 2 (ACTA2) was observed in peripubertal and adult KS as well as in Sertoli cell only (SCO) patients. Altered expression patterns for all ECM proteins were observed in SCO and KS biopsy tissues compared with controls. Only for collagen I and IV were altered expression patterns observed between KS and SCO patients. In peripubertal samples, no statistically significant differences were observed in the maturation markers, but altered ECM patterns were already present in some samples. CONCLUSION(S): The role of loss of ACTA2 expression in PTMC in the disintegration of tubules in KS patients should be further investigated. Future research is necessary to identify the causes of testicular fibrosis in KS patients. If the mechanism behind this fibrotic process could be identified, this process might be altered toward increasing the chances of fertility in KS patients.

Fertility Preservation in Childhood Cancer: Endocrine Activity in Prepubertal Human Testis Xenografts Exposed to a Pubertal Hormone Environment.

Survivors of childhood cancer are at risk for long-term treatment-induced health sequelae, including gonadotoxicity and iatrogenic infertility. At present, for prepubertal boys there are no viable clinical options to preserve future reproductive potential. We investigated the effect of a pubertal induction regimen with gonadotrophins on prepubertal human testis xenograft development. Human testis tissue was obtained from patients with cancer and non-malignant haematological disorders (n = 6; aged 1-14 years) who underwent testis tissue cryopreservation for fertility preservation. Fresh and frozen-thawed testis fragments were transplanted subcutaneously or intratesticularly into immunocompromised mice. Graft-bearing mice received injections of vehicle or exogenous gonadotrophins, human chorionic gonadotrophin (hCG, 20 IU), and follicle-stimulating hormone (FSH, 12.5 IU) three times a week for 12 weeks. The gross morphology of vehicle and gonadotrophin-exposed grafts was similar for both transplantation sites. Exposure of prepubertal human testis tissue xenografts to exogenous gonadotrophins resulted in limited endocrine function of grafts, as demonstrated by the occasional expression of the steroidogenic cholesterol side-chain cleavage enzyme (CYP11A1). Plasma testosterone concentrations (0.13 vs. 0.25 ng/mL; p = 0.594) and seminal vesicle weights (10.02 vs. 13.93 mg; p = 0.431) in gonadotrophin-exposed recipient mice were comparable to vehicle-exposed controls. Regardless of the transplantation site and treatment, initiation and maintenance of androgen receptor (AR) expression were observed in Sertoli cells, indicating commitment towards a more differentiated status. However, neither exogenous gonadotrophins (in castrated host mice) nor endogenous testosterone (in intact host mice) were sufficient to repress the expression of markers associated with immature Sertoli cells, such as anti-Müllerian hormone (AMH) and Ki67, or to induce the redistribution of junctional proteins (connexin 43, CX43; claudin 11, CLDN11) to areas adjacent to the basement membrane. Spermatogonia did not progress developmentally but remained the most advanced germ cell type in testis xenografts. Overall, these findings demonstrate that exogenous gonadotrophins promote partial activation and maturation of the somatic environment in prepubertal testis xenografts. However, alternative hormone regimens or additional factors for pubertal induction are required to complete the functional maturation of the spermatogonial stem cell (SSC) niche.

Does co-transplantation of mesenchymal and spermatogonial stem cells improve reproductive efficiency and safety in mice?

BACKGROUND: Spermatogonial stem cell transplantation (SSCT) is a promising therapy in restoring the fertility of childhood cancer survivors. However, the low efficiency of SSCT is a significant concern. SSCT could be improved by co-transplanting transforming growth factor beta 1 (TGFß1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFß1-induced MSCs. METHODS: A mouse model for long-term infertility was used to transplant SSCs (SSCT, n = 10) and a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). Both transplanted groups and a fertile control group (n = 7) were allowed to mate naturally to check the reproductive efficiency after transplantation. Furthermore, the testes from transplanted males and donor-derived male offspring were analyzed for the epigenetic markers DNA methyltransferase 3A (DNMT3A) and histone 4 lysine 5 acetylation (H4K5ac). RESULTS: The overall tubular fertility index (TFI) after SSCT (76 ± 12) was similar to that after MSi-SSCT (73 ± 14). However, the donor-derived TFI after MSi-SSCT (26 ± 14) was higher compared to the one after SSCT (9 ± 5; P = 0.002), even after injecting half of the number of SSCs in MSi-SSCT. The litter sizes after SSCT (3.7 ± 3.7) and MSi-SSCT (3.7 ± 3.6) were similar but differed significantly with the control group (7.6 ± 1.0; P < 0.001). The number of GFP+ offspring per litter obtained after SSCT (1.6 ± 0.5) and MSi-SSCT (2.0 ± 1.0) was also similar. The expression of DNMT3A and H4K5ac in germ cells of transplanted males was found to be significantly reduced compared to the control group. However, in donor-derived offspring, DNMT3A and H4K5ac followed the normal pattern. CONCLUSION: Co-transplanting SSCs and TGFß1-treated MSCs results in reproductive efficiency as good as SSCT, even after transplanting half the number of SSCs. Although transplanted males showed lower expression of DNMT3A and H4K5ac in donor-derived germ cells, the expression was restored to normal levels in germ cells of donor-derived offspring. This procedure could become an efficient method to restore fertility in a clinical setup, but more studies are needed to ensure safety in the long term.

Cryopreservation of Human Testicular Tissue by Isopropyl-Controlled Slow Freezing.

Tissue cryopreservation uses very low temperatures to preserve structurally intact living cells in their natural microenvironment. Cell survival is strongly influenced by the biophysical effects of ice during both the freezing and the subsequent thawing. These effects can be controlled by optimizing the fragment size, type of cryoprotectant, and cooling rate. The challenge is to determine cryopreservation parameters that suit all cell types present in the tissue. Here we describe a quick and convenient protocol for the cryopreservation of testicular tissue using an isopropyl-insulated freezing device, which was validated in both a mouse and a human model.

Co-transplantation of mesenchymal stem cells improves spermatogonial stem cell transplantation efficiency in mice.

BACKGROUND: Spermatogonial stem cell transplantation (SSCT) could become a fertility restoration tool for childhood cancer survivors. However, since in mice, the colonization efficiency of transplanted spermatogonial stem cells (SSCs) is only 12%, the efficiency of the procedure needs to be improved before clinical implementation is possible. Co-transplantation of mesenchymal stem cells (MSCs) might increase colonization efficiency of SSCs by restoring the SSC niche after gonadotoxic treatment. METHODS: A mouse model for long-term infertility was developed and used to transplant SSCs (SSCT, n = 10), MSCs (MSCT, n = 10), a combination of SSCs and MSCs (MS-SSCT, n = 10), or a combination of SSCs and TGFß1-treated MSCs (MSi-SSCT, n = 10). RESULTS: The best model for transplantation was obtained after intraperitoneal injection of busulfan (40 mg/kg body weight) at 4 weeks followed by CdCl2 (2 mg/kg body weight) at 8 weeks of age and transplantation at 11 weeks of age. Three months after transplantation, spermatogenesis resumed with a significantly better tubular fertility index (TFI) in all transplanted groups compared to non-transplanted controls (P < 0.001). TFI after MSi-SSCT (83.3 ± 19.5%) was significantly higher compared to MS-SSCT (71.5 ± 21.7%, P = 0.036) but did not differ statistically compared to SSCT (78.2 ± 12.5%). In contrast, TFI after MSCT (50.2 ± 22.5%) was significantly lower compared to SSCT (P < 0.001). Interestingly, donor-derived TFI was found to be significantly improved after MSi-SSCT (18.8 ± 8.0%) compared to SSCT (1.9 ± 1.1%; P < 0.001), MSCT (0.0 ± 0.0%; P < 0.001), and MS-SSCT (3.4 ± 1.9%; P < 0.001). While analyses showed that both native and TGFß1-treated MSCs maintained characteristics of MSCs, the latter showed less migratory characteristics and was not detected in other organs. CONCLUSION: Co-transplanting SSCs and TGFß1-treated MSCs significantly improves the recovery of endogenous SSCs and increases the homing efficiency of transplanted SSCs. This procedure could become an efficient method to treat infertility in a clinical setup, once the safety of the technique has been proven.

Does early cell death cause germ cell loss after intratesticular tissue grafting?

OBJECTIVE: To assess cell death in intratesticular grafts. DESIGN: Experimental animal study. SETTING: University. ANIMAL(S): F1-hybrids from SV129 X C57BL. INTERVENTION(S): Intratesticular tissue transplantation was performed and cell death in the grafts was evaluated at different time points after transplantation. MAIN OUTCOME MEASURE(S): Apoptotic cell death in spermatogonia was evaluated by flow cytometry with the use of the annexin V assay. Immunohistochemistry was used to evaluate graft development and the global occurrence of cell death. RESULT(S): The highest level of spermatogonia-specific cell death was found on days 4 and 10, although no statistical difference was observed compared with control tissue. Statistically significant reductions in tubule integrity were observed 1 day and 2 months after transplantation. More degenerated tubules were observed in the center of the grafts 1 and 4 days after transplantation, and higher numbers of apoptotic tubules were found 1 day after transplantation. No difference in overall cell death was observed between grafts and controls for any time point except for the frozen grafts 1 day after transplantation. CONCLUSION(S): Spermatogonia-specific apoptosis does not explain the stem cell loss observed after intratesticular tissue grafting; it probably results from degeneration of tubules in the center of the graft owing to hypoxia during the first days after transplantation.

Presence of spermatogonia in 47,XXY men with no spermatozoa recovered after testicular sperm extraction.

OBJECTIVE: To evaluate the presence of spermatogonia in men diagnosed with Klinefelter syndrome (KS), in whom no testicular spermatozoa were recovered by testicular sperm extraction. DESIGN: Retrospective case series. SETTING: University hospital. PATIENT(S): Testicular samples from 22 nonmosaic 47,XXY men, aged 24-43 years, with no spermatozoa at multiple biopsies. INTERVENTION(S): Paraffin-embedded testicular tissue was sectioned and stained with hematoxylin-eosin, and immunostainings were performed for both MAGE-A4 and vimentin. MAIN OUTCOME MEASURE(S): The presence of spermatogonia. RESULT(S): Massive fibrosis and hyalinization were observed in all men with KS. Spermatogonia were observed in 4 of 22 men with KS, with differentiation up to the spermatocyte level in 2 of them. CONCLUSION(S): A few men with KS, having no spermatozoa after testicular sperm extraction, still had few spermatogonia. These patients may eventually benefit from in vitro maturation using spermatogonial stem cells. The adult KS population can thus be divided into three subgroups: one subgroup showing focal spermatogenesis, a second having spermatogonia, and a third group in which no germ cells can be recovered. Further research is necessary to unravel the mechanism leading to these different patterns in patients with KS.

Spermatogonial stem cell preservation in boys with Klinefelter syndrome: to bank or not to bank, that's the question.

Although early development of testis appears normal in boys with Klinefelter syndrome (KS), spermatogonial stem cell (SSC) depletion occurs in midpuberty, leading to infertility. Therefore, freezing of semen samples or testicular tissue sampling could be offered to boys with KS at onset of puberty. However, only in about half of patients with KS, adult or prepubertal, spermatozoa or SSCs can be observed, and to date, no clinical parameters are available to detect patients who might benefit from these techniques. Furthermore, strategies for the further use of the cryopreserved material are still under investigation. Retrieval of spermatogonial cells in prepubertal boys with KS should therefore still be viewed as experimental and patients and their parents must be counseled accordingly.

Orthotopic grafting of cryopreserved prepubertal testicular tissue: in search of a simple yet effective cryopreservation protocol.

OBJECTIVE: To investigate whether solid-surface vitrification (SSV) is an effective cryopreservation strategy regarding the integrity and function of prepubertal mouse testicular tissue. DESIGN: Prospective experimental study. SETTING: Academic research unit. ANIMAL(S): Mice. INTERVENTION(S): Testicular tissue from 5- to 10-day-old GFP(+) mice was cryopreserved with the use of a conventional uncontrolled slow freezing (USF) technique and SSV before intratesticular grafting in busulfan-treated GFP(-) mice. MAIN OUTCOME MEASURE(S): Ultrastructural cryoinjury to spermatogonial stem cells (SSCs) and somatic cells was assessed by electron microscopy. Tubular structure was evaluated by histology, and graft survival and spermatogenic recovery by immunohistochemistry. RESULT(S): The tubular morphology and the proportion of ultrastructural cryodamage were similar between vitrified and slow-frozen testicular fragments. Allografting of tissue after both USF and SSV resulted in a recovery of spermatogenesis similar to fresh samples. CONCLUSION(S): SSV resulted in success rates similar to USF in maintaining testicular cell ultrastructure, tubular morphology, and tissue function. These data provide further evidence that vitrification, being an inexpensive and simple technique, can be considered as an alternative for cryopreservation of prepubertal testicular tissue.

Regeneration of spermatogenesis by grafting testicular tissue or injecting testicular cells into the testes of sterile mice: a comparative study.

OBJECTIVE: To make a comparison between two different approaches-spermatogonial stem cell transplantation and intratesticular grafting, for preservation and reintroduction of spermatogonial stem cells. DESIGN: Prospective experimental study. SETTING: Academic medical center and teaching hospital. PATIENT(S): N/A. INTERVENTION(S): Intratesticular transplantation, histologic evaluation of testes. MAIN OUTCOME MEASURE(S): Testicular weight, amount of green fluorescence in the testis, and immunostaining for green fluorescent protein. RESULT(S): In a first experiment donor-derived spermatogenesis was found in 65% of the injected testes (41.8 +/- 72.2 mm) compared with 75% of the testes (122.1 +/- 45.6 mm) after tissue grafting. In the second series of experiments complete spermatogenesis was found in 75% of the testes after fresh grafting (93.8 +/- 21.8 mm) compared with 88% after frozen-thawed tissue grafting (84.8 +/- 45.6 mm). CONCLUSION(S): Both approaches show that spermatogonial stem cells can successfully be introduced to the testis resulting in spermatogenesis. Tissue grafting produced a larger mean donor colony length and there was no significant difference between colonization efficiency using either fresh or frozen-thawed grafts. In a future clinical setting, grafting would be a simple and efficient way for reintroducing stem cells to the testis.

Bone marrow stem cells transplanted to the testis of sterile mice do not differentiate into spermatogonial stem cells and have no protective effect on fertility.

Four months after transplanting bone marrow cells into the testis, no differentiation to spermatogonial stem cells was observed. Bone marrow transplantation had no protective effect on fertility after chemotherapy.