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This study presents a biphasic approach to overcome the limitations of current testicular organoid (TO) cultures, including histological heterogeneity, germ cell loss and absence of spermatogenesis. Agarose microwells were utilized to create TOs from prepubertal C57BL/6 J testicular cells. First emphasis was on improving germ cell survival during the initial 2-week reorganization phase by comparingα-MEM + 10% knockout serum replacement (KSR) medium, known to support TO generation in mice, to three optimized media (1-3). Cell densities and culture dynamics were also tested to recreate histological resemblance to testes. After optimizing germ cell survival and cell organization, the effect of growth factors and immunomodulation through CD45+immune cell depletion or dexamethasone (DEX) supplementation were assessed for enhancing spermatogenesis during the subsequent differentiation phase. Testicular cells self-reorganized into organoids resembling the testicular anatomical unit, characterized by one tubule-like structure surrounded by interstitium. Media 1-3 proved superior for organoid growth during the reorganization phase, with TOs in medium 3 exhibiting germ cell numbers (7.4% ± 4.8%) comparable to controls (9.3% ± 5.3%). Additionally, 37% ± 30% demonstrated organized histology from 32 × 103cells under static conditions. Switching toα-MEM + 10% KSR during the differentiation phase increased formation efficiency to 85 ± 7%, along with elevated germ cell numbers, testosterone production (3.1 ± 0.9 ng ml-1) and generation ofγ-H2AX+spermatid-like cells (steps 8-11, 1.2% ± 2.2% of the total). Adding differentiation factors to theα-MEM increased spermatid-like cell numbers to 2.9% ± 5.9%, confirmed through positive staining for CREM, transition protein 1, and peanut agglutinin. Although, these remained diploid with irregular nuclear maturation. DEX supplementation had no additional effect, and immune cell depletion adversely impacted TO formation. The manipulability of TOs offers advantages in studying male infertility and exploring therapies, with scalability enabling high-throughput chemical screening and reducing animal usage in reproductive toxicity and drug discovery studies.
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Supervivencia Celular , Ratones Endogámicos C57BL , Organoides , Espermatogénesis , Testículo , Testosterona , Masculino , Animales , Organoides/citología , Organoides/metabolismo , Organoides/efectos de los fármacos , Testículo/citología , Testículo/efectos de los fármacos , Testículo/metabolismo , Testosterona/farmacología , Espermatogénesis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ratones , Diferenciación Celular/efectos de los fármacos , Células Germinativas/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/metabolismo , Dexametasona/farmacologíaRESUMEN
Klinefelter syndrome (47,XXY) causes infertility with a testicular histology comprising two types of Sertoli cell-only tubules, representing mature and immature-like Sertoli cells, and occasionally focal spermatogenesis. Here, we show that the immature-like Sertoli cells highly expressed XIST and had two X-chromosomes, while the mature Sertoli cells lacked XIST expression and had only one X-chromosome. Sertoli cells supporting focal spermatogenesis also lacked XIST expression and the additional X-chromosome, while the spermatogonia expressed XIST despite having only one X-chromosome. XIST was expressed in Sertoli cells until puberty, where a gradual loss was observed. Our results suggest that a micro-mosaic loss of the additional X-chromosome is needed for Sertoli cells to mature and to allow focal spermatogenesis.
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Síndrome de Klinefelter , ARN Largo no Codificante , Células de Sertoli , Espermatogénesis , Síndrome de Klinefelter/genética , Síndrome de Klinefelter/patología , Síndrome de Klinefelter/metabolismo , Masculino , Células de Sertoli/metabolismo , Células de Sertoli/patología , Espermatogénesis/genética , Animales , Humanos , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromosomas Humanos X/genética , Cromosoma X/genéticaRESUMEN
In recent years, immune checkpoint inhibitors (ICIs) have become a viable option for many cancer patients, including specific subgroups of pediatric patients. Despite their efficiency in treating different types of cancer, ICIs are responsible for a number of immune-related adverse events, including inflammatory toxicities, that can affect several organs. However, our knowledge of the impact of ICIs on the testis and male fertility is limited. It is possible that ICI treatment affects testicular function and spermatogenesis either directly or indirectly (or both). Treatment with ICIs may cause increased inflammation and immune cell infiltration within the seminiferous tubules of the testis, disturbing spermatogenesis or testosterone deficiency (primary hypogonadism). Additionally, the interference of ICIs with the hypothalamic-pituitary-gonadal axis may alter testosterone production, affecting testicular function (secondary hypogonadism) and spermatogenesis. This review provides an overview of the available evidence on the potential association between ICIs and the disruption of spermatogenesis, with special focus on ICIs targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1). Moreover, it highlights the need for further investigations and encourages the discussion of associated risks and fertility-preservation considerations between clinicians and patients.
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STUDY QUESTION: Twenty years after the inception of the first fertility preservation programme for pre-pubertal boys, what are the current international practices with regard to cryopreservation of immature testicular tissue? SUMMARY ANSWER: Worldwide, testicular tissue has been cryopreserved from over 3000 boys under the age of 18 years for a variety of malignant and non-malignant indications; there is variability in practices related to eligibility, clinical assessment, storage, and funding. WHAT IS KNOWN ALREADY: For male patients receiving gonadotoxic treatment prior to puberty, testicular tissue cryopreservation may provide a method of fertility preservation. While this technique remains experimental, an increasing number of centres worldwide are cryopreserving immature testicular tissue and are approaching clinical application of methods to use this stored tissue to restore fertility. As such, standards for quality assurance and clinical care in preserving immature testicular tissue should be established. STUDY DESIGN SIZE DURATION: A detailed survey was sent to 17 centres within the recently established ORCHID-NET consortium, which offer testicular tissue cryopreservation to patients under the age of 18 years. The study encompassed 60 questions and remained open from 1 July to 1 November 2022. PARTICIPANTS/MATERIALS SETTING METHODS: Of the 17 invited centres, 16 completed the survey, with representation from Europe, Australia, and the USA. Collectively, these centres have cryopreserved testicular tissue from patients under the age of 18 years. Data are presented using descriptive analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Since the establishment of the first formal fertility preservation programme for pre-pubertal males in 2002, these 16 centres have cryopreserved tissue from 3118 patients under the age of 18 years, with both malignant (60.4%) and non-malignant (39.6%) diagnoses. All centres perform unilateral biopsies, while 6/16 sometimes perform bilateral biopsies. When cryopreserving tissue, 9/16 centres preserve fragments sized ≤5 mm3 with the remainder preserving fragments sized 6-20 mm3. Dimethylsulphoxide is commonly used as a cryoprotectant, with medium supplements varying across centres. There are variations in funding source, storage duration, and follow-up practice. Research, with consent, is conducted on stored tissue in 13/16 centres. LIMITATIONS REASONS FOR CAUTION: While this is a multi-national study, it will not encompass every centre worldwide that is cryopreserving testicular tissue from males under 18 years of age. As such, it is likely that the actual number of patients is even higher than we report. Whilst the study is likely to reflect global practice overall, it will not provide a complete picture of practices in every centre. WIDER IMPLICATIONS OF THE FINDINGS: Given the research advances, it is reasonable to suggest that cryopreserved immature testicular tissue will in the future be used clinically to restore fertility. The growing number of patients undergoing this procedure necessitates collaboration between centres to better harmonize clinical and research protocols evaluating tissue function and clinical outcomes in these patients. STUDY FUNDING/COMPETING INTERESTS: K.D. is supported by a CRUK grant (C157/A25193). R.T.M. is supported by an UK Research and Innovation (UKRI) Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health at the University of Edinburgh is supported by MRC (MR/N022556/1). C.L.M. is funded by Kika86 and ZonMW TAS 116003002. A.M.M.v.P. is supported by ZonMW TAS 116003002. E.G. was supported by the Research Program of the Research Foundation-Flanders (G.0109.18N), Kom op tegen Kanker, the Strategic Research Program (VUB_SRP89), and the Scientific Fund Willy Gepts. J.-B.S. is supported by the Swedish Childhood Cancer Foundation (TJ2020-0026). The work of NORDFERTIL is supported by the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115), the Swedish Research Council (2018-03094; 2021-02107), and the Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2021-00073; 2022-00317; 2023-00353). C.E is supported by the Health Department of the Basque Government (Grants 2019111068 and 2022111067) and Inocente Inocente Foundation (FII22/001). M.P.R. is funded by a Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1. A.F. and N.R. received support from a French national research grant PHRC No. 2008/071/HP obtained by the French Institute of Cancer and the French Healthcare Organization. K.E.O. is funded by the University of Pittsburgh Medical Center and the US National Institutes of Health HD100197. V.B-L is supported by the French National Institute of Cancer (Grant Seq21-026). Y.J. is supported by the Royal Children's Hospital Foundation and a Medical Research Future Fund MRFAR000308. E.G., N.N., S.S., C.L.M., A.M.M.v.P., C.E., R.T.M., K.D., M.P.R. are members of COST Action CA20119 (ANDRONET) supported by COST (European Cooperation in Science and Technology). The Danish Child Cancer Foundation is also thanked for financial support (C.Y.A.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.
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Nursing students are confronted with euthanasia during their internship and certainly during their later career but they feel inadequately prepared in dealing with a euthanasia request. This study presents a simulation module focused on euthanasia and evaluates nursing students' perceptions after they have completed the simulation practice. The 'euthanasia module' consisted of a preparatory online learning module, a good-practice video, an in-vivo simulation scenario, and a debriefing session. The module's content was validated by four experts in end-of-life care. The module was completed by three groups of students from two different University Colleges (n = 17 in total). The students were divided into three groups: one with no previous simulation education experience, one with all students having previous experience, and another with a mix of experiences. After completing the entire module, each group had a discussion regarding their perceptions and expectations concerning this simulation module. Thematic content analysis was conducted on audio-recorded group interviews using NVIVO® software, involving initial open coding, transformation into specific themes and subthemes through axial coding, and defining core themes through selective coding, with data analysis continuing until data saturation was reached. Students generally found the online learning module valuable for background information, had mixed perceptions of the good-practice video, and appreciated the well-crafted scenarios with the taboo of euthanasia emerging during simulations, while the debriefing process was seen as enhancing clinical reasoning abilities. Students considered the simulation module a valuable addition to their education and nursing careers, expressing their satisfaction with the comprehensive coverage of the sensitive topic presented without sensationalism or taboos. This subject holds significance for nations with established euthanasia laws and those lacking such legal frameworks. The findings of this study can aid teachers in developing and accessing euthanasia simulation training programs, contributing to broader education's emphasis on integrating euthanasia-related knowledge and skills.
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Bachillerato en Enfermería , Eutanasia , Entrenamiento Simulado , Estudiantes de Enfermería , Humanos , Satisfacción Personal , Competencia ClínicaRESUMEN
Testes have a complex architecture that is compartmentalized into seminiferous tubules with a diameter of approximatively 200 µm in which the germ cells differentiate, surrounded by a basement membrane and interstitium. 3D bioprinting might be used to recreate the compartmentalized testicular architecture in vitro. Directed by a software program, pneumatic microextrusion printers can deposit 3D layers of hydrogel-encapsulated interstitial cells in a controlled manner by applying pressure. Once macroporous-shaped scaffolds resembling seminiferous tubules have been bioprinted with interstitial cells, the epithelial cell fraction can be seeded in the macropores to resemble the in vivo testicular architecture. Moreover, macropores can serve as a delimitation for all testicular cells to reorganize and improve the supply of nutrients to cells through the 3D constructs.
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Bioimpresión , Espermatogénesis , Masculino , Animales , Ratones , Testículo , Túbulos Seminíferos , Andamios del Tejido , Células Intersticiales del Testículo , Hidrogeles , Ingeniería de Tejidos , Impresión TridimensionalRESUMEN
BACKGROUND: Experimental fertility preservation programs have been started to safeguard the future fertility of prepubertal and pubertal males requiring high-risk gonadotoxic treatment protocols. However, long-term follow-up studies evaluating the effects on their gonadal development and function related to the testicular biopsy procedure are rather limited. DESIGN: This two-center follow-up study (between 2002 and 2020) evaluated the gonadal development and function of a cohort of 59 prepubertal and pubertal males who have been offered immature testicular tissue banking (TTB) prior to conventional high-risk chemo- and/or radiotherapy (HR-C/R) or conditioning therapy before hematopoietic stem cell transplantation (CT-HSCT). The aim is to investigate the long-term impact of the testicular biopsy procedure and the high-risk gonadotoxic treatment. Testicular growth and the reproductive hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone (T), and inhibin B (INHB) were analyzed after treatment completion, and compared between males accepting TTB and those refusing TTB (control) as well as between HR-C/R and CT-HSCT treatment protocols. RESULTS: Of the 59 prepubertal and pubertal males included, 25 were treated by HR-C/R and 34 required CT-HSCT. TTB was accepted for 39 males and refused for 20 males. Most patients were prepubertal at diagnosis (85%), at TTB (79%), and at treatment completion (76%), and pubertal or postpubertal at their last follow-up visit (66%). After 5.0 (1.0-13.0) years post treatment, most patients show normal testicular volumes (83%) and normal LH (89%), FSH (87%), T (87%), and INHB (79%) serum levels. The testicular biopsy procedure did not have an effect on testicular growth, LH, FSH, T, and INHB. Significantly more small postpubertal testicular volumes (p = .0278) and low INHB serum levels (p = .0130) were recorded after CT-HSCT, especially after myeloablative conditioning. CONCLUSION: The clinical follow-up data demonstrate no effect related to the biopsy procedure, but a substantial risk for impaired gonadal development after high-risk gonadotoxic treatment, in particular myeloablative CT-HSCT. Longer follow-up studies with a larger study population are needed to confirm these preliminary findings.
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Hormona Luteinizante , Testículo , Masculino , Humanos , Estudios de Seguimiento , Hormona Folículo Estimulante , TestosteronaRESUMEN
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.
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STUDY QUESTION: What is the impact of cancer or hematological disorders on germ cells in pediatric male patients? SUMMARY ANSWER: Spermatogonial quantity is reduced in testes of prepubertal boys diagnosed with cancer or severe hematological disorder compared to healthy controls and this reduction is disease and age dependent: patients with central nervous system cancer (CNS tumors) and hematological disorders, as well as boys <7 years are the most affected. WHAT IS KNOWN ALREADY: Fertility preservation in pediatric male patients is considered based on the gonadotoxicity of selected treatments. Although treatment effects on germ cells have been extensively investigated, limited data are available on the effect of the disease on the prepubertal male gonad. Of the few studies investigating the effects of cancer or hematologic disorders on testicular function and germ cell quantity in prepuberty, the results are inconsistent. However, recent studies suggested impairments before the initiation of known gonadotoxic therapy. Understanding which diseases and at what age affect the germ cell pool in pediatric patients before treatment is critical to optimize strategies and counseling for fertility preservation. STUDY DESIGN, SIZE, DURATION: This multicenter retrospective cohort study included 101 boys aged <14 years with extra-cerebral cancer (solid tumors), CNS tumors, leukemia/lymphoma (blood cancer), or non-malignant hematological disorders, who were admitted for a fertility preservation programme between 2002 and 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: In addition to clinical data, we analyzed measurements of testicular volume and performed histological staining on testicular biopsies obtained before treatment, at cryopreservation, to evaluate number of spermatogonia per tubular cross-section, tubular fertility index, and the most advanced germ cell type prior to chemo-/radiotherapy. The controls were data simulations with summary statistics from original studies reporting healthy prepubertal boys' testes characteristics. MAIN RESULTS AND THE ROLE OF CHANCE: Prepubertal patients with childhood cancer or hematological disorders were more likely to have significantly reduced spermatogonial quantity compared to healthy controls (48.5% versus 31.0% prevalence, respectively). The prevalence of patients with reduced spermatogonial quantity was highest in the CNS tumor (56.7%) and the hematological disorder (55.6%) groups, including patients with hydroxyurea pre-treated sickle cell disease (58.3%) and patients not exposed to hydroxyurea (50%). Disease also adversely impacted spermatogonial distribution and differentiation. Irrespective of disease, we observed the highest spermatogonial quantity reduction in patients <7 years of age. LIMITATIONS, REASONS FOR CAUTION: For ethical reasons, we could not collect spermatogonial quantity data in healthy prepubertal boys as controls and thus deployed statistical simulation on data from literature. Also, our results should be interpreted considering low patient numbers per (sub)group. WIDER IMPLICATIONS OF THE FINDINGS: Cancers, especially CNS tumors, and severe hematological disorders can affect spermatogonial quantity in prepubertal boys before treatment. Consequently, these patients may have a higher risk of depleted spermatogonia following therapies, resulting in persistent infertility. Therefore, patient counseling prior to disease treatment and timing of fertility preservation should not only be based on treatment regimes, but also on diagnoses and age. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Marie Curie Initial Training Network (ITN) (EU-FP7-PEOPLE-2013-ITN) funded by European Commision grant no. 603568; ZonMW Translational Adult stem cell research (TAS) grant no. 116003002. No competing interests. TRIAL REGISTRATION NUMBER: N/A.
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Preservación de la Fertilidad , Enfermedades Hematológicas , Neoplasias , Adulto , Niño , Humanos , Masculino , Espermatogonias , Preservación de la Fertilidad/métodos , Estudios Retrospectivos , Hidroxiurea , Testículo , CriopreservaciónRESUMEN
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.
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Síndrome de Klinefelter , Masculino , Humanos , Síndrome de Klinefelter/patología , Transcriptoma , Testículo/metabolismo , Fibrosis , ARN/metabolismo , Atrofia/patologíaRESUMEN
Gene editing in male germline stem (GS) cells is a potent tool to study spermatogenesis and to create transgenic mice. Various engineered nucleases already demonstrated the ability to modify the genome of GS cells. However, current systems are limited by technical complexity diminishing application options. To establish an easier method to mediate gene editing, we tested the lipofection of site-specific Cas9:gRNA ribonucleoprotein (RNP) complexes to knockout the enhanced green fluorescent protein (Egfp) in mouse EGFP-GS cells via non-homologous end joining. To monitor whether gene conversion through homology-directed repair events occurred, single-stranded oligodeoxynucleotides were co-lipofected to deliver a Bfp donor sequence. Results showed Egfp knockout in up to 22% of GS cells, which retained their undifferentiated status following transfection, while only less than 0.7% EGFP to BFP conversion was detected in gated GS cells. These data show that CRISPR/Cas9 RNP-based lipofection is a promising system to simply and effectively knock out genes in mouse GS cells. Understanding the genes involved in spermatogenesis could expand therapeutic opportunities for men suffering from infertility.
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Due to the growing number of young patients at risk of germ cell loss, there is a need to preserve spermatogonial stem cells for patients who are not able to bank spermatozoa. Worldwide, more and more clinics are implementing testicular tissue (TT) banking programs, making it a novel, yet indispensable, discipline in the field of fertility preservation. Previously, TT cryopreservation was predominantly offered to young cancer patients before starting gonadotoxic chemo- or radiotherapy. Nowadays, most centers also bank TT from patients with non-malignant conditions who need gonadotoxic conditioning therapy prior to hematopoietic stem cell (HSCT) or bone marrow transplantation (BMT). Additionally, some centers include patients who suffer from genetic or developmental disorders associated with prepubertal germ cell loss or patients who already had a previous round of chemo- or radiotherapy. It is important to note that the surgical removal of TT is an invasive procedure. Moreover, TT cryopreservation is still considered experimental as restoration methods are not yet clinically available. For this reason, TT banking should preferably only be offered to patients who are at significant risk of becoming infertile. In our view, TT cryopreservation is recommended for young cancer patients in need of high-risk chemo- and/or radiotherapy, regardless of previous low-risk treatment. Likewise, TT banking is advised for patients with non-malignant disorders such as sickle cell disease, beta-thalassemia, and bone marrow failure, who need high-risk conditioning therapy before HSCT/BMT. TT retrieval during orchidopexy is also proposed for patients with bilateral cryptorchidism. Since patients with a medium- to low-risk treatment generally maintain their fertility, TT banking is not advised for this group. Also for Klinefelter patients, TT banking is not recommended as it does not give better outcomes than a testicular sperm extraction later in life.
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Preservación de la Fertilidad , Criopreservación/métodos , Preservación de la Fertilidad/métodos , Humanos , Masculino , Espermatozoides , Testículo , Bancos de TejidosRESUMEN
RESEARCH QUESTION: Which early-diagnosed Klinefelter syndrome patients have been offered cryopreservation of testicular tissue as part of fertility preservation before spermatogonial stem cell (SSC) loss? Do these Klinefelter syndrome patients present with behavioural, cognitive and/or psychological problems? Does a testicular biopsy procedure have long-term effects on the gonadal development of Klinefelter syndrome patients? DESIGN: Early-diagnosed Klinefelter syndrome patients followed between 2009 and 2020 and offered testicular tissue banking in an experimental context at the Universitair Ziekenhuis Brussel were included. The prevalence of behavioural, cognitive and/or psychological problems was determined. Changes in testicular volume and in gonadal function (LH, FSH, testosterone and inhibin B [INHB]) were studied. RESULTS: Of the 48 Klinefelter syndrome patients included, 22 had testicular tissue removed (biopsy group) and 26 had no surgical intervention (control group). The need for specialized education was significantly higher in prenatally (P = 0.0159) and prepubertally (P = 0.0002) diagnosed Klinefelter syndrome patients. Psychological problems were significantly more prevalent in Klinefelter syndrome patients who did not opt for fertility preservation (P = 0.0447). In the first 4.2 (1.9-9.1) years after testicular biopsy, no difference in testicular volume was observed between the biopsied and the contralateral non-biopsied testes (P > 0.9999). After pubertal onset, no differences in LH, FSH, testosterone and INHB were found between the biopsy and the control groups (P = 0.1324 for LH, P > 0.9999 for FSH, P = 0.5433 for testosterone, P > 0.9999 for INHB). CONCLUSION: Early-diagnosed Klinefelter syndrome patients presented with behavioural, cognitive and/or psychological problems. Only psychological problems seemed to influence the decision towards fertility preservation. Follow-up data confirm that harvesting testicular tissue does not have a long-term impact on the gonadal development of Klinefelter syndrome patients.
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Preservación de la Fertilidad , Síndrome de Klinefelter , Biopsia , Femenino , Preservación de la Fertilidad/métodos , Hormona Folículo Estimulante , Estudios de Seguimiento , Humanos , Síndrome de Klinefelter/complicaciones , Síndrome de Klinefelter/diagnóstico , Síndrome de Klinefelter/patología , Masculino , Testículo/patología , TestosteronaRESUMEN
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 (nâ¯=â¯3) and controls (nâ¯=â¯2) (<18 years) was grafted to the mouse testis (nâ¯=â¯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 (nâ¯=â¯5) and controls (nâ¯=â¯3), transplanted to mice (nâ¯=â¯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.
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Síndrome de Klinefelter , Testículo , Animales , Femenino , Fibrosis , Humanos , Cetotifen , Síndrome de Klinefelter/patología , Estudios Longitudinales , Masculino , Ratones , Espermatogénesis , Espermatogonias , Testículo/patología , Trasplante HeterólogoRESUMEN
Cystinosis is an inherited metabolic disorder caused by autosomal recessive mutations in the CTNS gene leading to lysosomal cystine accumulation. The disease primarily affects the kidneys followed by extra-renal organ involvement later in life. Azoospermia is one of the unclarified complications which are not improved by cysteamine, which is the only available disease-modifying treatment. We aimed at unraveling the origin of azoospermia in cysteamine-treated cystinosis by confirming or excluding an obstructive factor, and investigating the effect of cysteamine on fertility in the Ctns-/- mouse model compared with wild type. Azoospermia was present in the vast majority of infantile type cystinosis patients. While spermatogenesis was intact, an enlarged caput epididymis and reduced levels of seminal markers for obstruction neutral α-glucosidase (NAG) and extracellular matrix protein 1 (ECM1) pointed towards an epididymal obstruction. Histopathological examination in human and mouse testis revealed a disturbed blood-testis barrier characterized by an altered zonula occludens-1 (ZO-1) protein expression. Animal studies ruled out a negative effect of cysteamine on fertility, but showed that cystine accumulation in the testis is irresponsive to regular cysteamine treatment. We conclude that the azoospermia in infantile cystinosis is due to an obstruction related to epididymal dysfunction, irrespective of the severity of an evolving primary hypogonadism. Regular cysteamine treatment does not affect fertility but has subtherapeutic effects on cystine accumulation in testis.
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Azoospermia/patología , Barrera Hematotesticular/metabolismo , Cisteamina/uso terapéutico , Cistinosis/tratamiento farmacológico , Testículo/patología , Adulto , Animales , Azoospermia/complicaciones , Azoospermia/genética , Depletores de Cistina/uso terapéutico , Cistinosis/complicaciones , Cistinosis/patología , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/genética , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Estudios Retrospectivos , Adulto Joven , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
Short-term germ cell survival and central tissue degeneration limit organoid cultures. Here, testicular organoids (TOs) were generated from two different mouse strains in 3D printed one-layer scaffolds (1LS) at the air-medium interface displaying tubule-like structures and Leydig cell functionality supporting long-term survival and differentiation of germ cells to the meiotic phase. Chimeric TOs, consisting of a mixture of primary testicular cells and EGFP+ germline stem (GS) cells, were cultured in two-layer scaffolds (2LSs) for better entrapment. They showed an improved spheroidal morphology consisting of one intact tubule-like structure and surrounding interstitium, representing the functional unit of a testis. However, GS cells did not survive long-term culture. Consequently, further optimization of the culture medium is required to enhance the maintenance and differentiation of germ cells. The opportunities TOs offer to manipulate somatic and germ cells are essential for the study of male infertility and the search for potential therapies.
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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.
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Personas Transgénero , Bélgica , Femenino , Humanos , Masculino , Estudios Prospectivos , Espermatogénesis , Espermatogonias , TestículoRESUMEN
Engineering of 3D substrates with maximum similarity to seminiferous tubules would help to produce functional sperm cells in vitro from stem cells. Here, we present a 3D electrospun gelatin (EG) substrate seeded with Sertoli cells and determine its potential for guided differentiation of embryonic stem cells (ESCs) toward germline cells. The EG was fabricated by electrospinning, and its morphology under SEM, as well as cytobiocompatibility for Sertoli cells and ESCs, was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and cell attachment assay. Embryoid bodies (EBs) were formed from ESCs and co-cultured with Sertoli cells, induced with BMP4 for 3 and 7 consecutive days to induce the differentiation of EBs toward germline cells. The differentiation was investigated by immunocytochemistry (ICC), flow cytometry, and RT-PCR in four experimental groups of EBs (EBs cultured in gelatin-coated cell culture plates); Scaffold/EB (EBs cultured on EG); ESCs/Ser (EBs and Sertoli cells co-cultured on gelatin-coated cell culture plates without EG); and Scaffold/EB/Ser (EBs and Sertoli cells co-cultured on EG). All experimental groups exhibited a significantly increased MVH (germline-specific marker) and decreased c-KIT (stemness marker) expression when compared with the EB group. ICC and flow cytometry revealed that Scaffold/EB/Ser had the highest level of MVH and the lowest c-KIT expression at both 3 and 7 days postdifferentiation compared with other groups. RT-PCR results showed a significant increase in the germline marker (Dazl) and a significant decrease in the ESC stemness marker (Nanog) in Scaffold/EB compared to the EB group. The germline markers Gcna, Stella, Mvh, Stra8, Piwil2, and Dazl were significantly increased in Scaffold/EB/Ser compared to the Scaffold/EB group. Our findings revealed that the EG scaffold can provide an excellent substrate biomimicking the micro/nanostructure of native seminiferous tubules and a platform for Sertoli cell-EB communication required for growth and differentiation of ESCs into germline cells.
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Células Madre Embrionarias , Gelatina , Células Cultivadas , Técnicas de Cocultivo , Masculino , EspermatozoidesRESUMEN
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.
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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.