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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.

As gonadotoxic adverse effects of antineoplastic treatments can result in infertility, gamete cryopreservation is routinely offered to patients as the strategy to preserve their fertility. However, there are many cases where gold standards cannot be applied, as is the case for prepubertal cancer patients and others unable to produce gametes or their precursors at the moment of diagnosis. With an increasing number of cancer survivors in our society, strategies using either cryopreserved gonadal tissue or stem cells have been developed to allow cancer survivors to achieve fatherhood, and recent advances in the field have increased public interest. In this review, we discuss the latest updates in fertility preservation from a basic and a clinical point of view. Fertility preservation for gonadotoxicity of antineoplastic therapies represents an important aspect of the quality of life of cancer survivors. Oocyte/embryo vitrification and sperm banking are the gold standards of fertility preservation. Ovarian tissue cryopreservation has already been employed to restore fertility in women. However, in vitro maturation of primordial follicles is still at the research stage in animals. Prepubertal testicular tissue cryopreservation and derived techniques to restore fertility in men have been successful in animal models but not yet in humans. The complete differentiation of iPSCs into gametes has been successful in mouse models but its low efficiency and the need to use a gonadal niche represents a barrier to its translation to humans. Transdifferentiation of somatic cells into germ cells may be a future alternative.

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 CdCl 2 (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.

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-β 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.

Young boys undergoing gonadotoxic treatments are at high risk of spermatogonial stem cell (SSC) loss and fertility problems later in life. Stem cell loss can also occur in specific genetic conditions, eg, Klinefelter syndrome (KS). Before puberty, these boys do not yet produce sperm. Hence, they cannot benefit from sperm banking. An emerging alternative is the freezing of testicular tissue aiming to preserve the SSCs for eventual autologous transplantation or in vitro maturation at adult age. Many fertility preservation programmes include cryopreservation of immature testicular tissue, although the restoration procedures are still under development. Until the end of 2018, the Universitair Ziekenhuis Brussel has frozen testicular tissues of 112 patients between 8 months and 18 years of age. Testicular tissue was removed in view of gonadotoxic cancer treatment (35%), gonadotoxic conditioning therapy for bone marrow transplantation (35%) or in boys diagnosed with KS (30%). So far, none of these boys had their testicular tissue transplanted back. This article summarizes our experience with cryopreservation of immature testicular tissue over the past 16 years (2002-2018) and describes the key issues for setting up a cryopreservation programme for immature testicular tissue as a means to safeguard the future fertility of boys at high risk of SSC loss.

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.

Previous studies have shown that the removal of one testis leads to a compensatory mechanism in the contralateral one, but this was species and age dependent. The aim of this study was to check whether this compensation would still occur after the combination of a unilateral orchiectomy and gonadotoxic treatment, since this resembles the clinical situation of patients who have to undergo highly toxic cancer treatment and therefore choose to cryopreserve a testicular biopsy for fertility restoration purposes. Sprague Dawley rats underwent either unilateral orchiectomy, gonadotoxic busulfan treatment, the combination of both or served as fertile control. A comparison of the compensatory effects was made between adult and prepubertal treated rats. Mating experiments were performed, testosterone levels were followed-up, testicular weight was recorded and histology was analysed. Adult treated rats were able to restore fertility spontaneously in all treatment groups. On the other hand, 30% of the rats that underwent a unilateral orchiectomy and gonadotoxic treatment at prepubertal age showed hampered spermatogenesis, low testosterone levels, decreased testicular weights and were not able to reproduce. This study emphasizes the need of fertility preservation strategies in prepubertal patients before gonadotoxic interventions.

Although the impact of gender-affirming hormone therapy (GAHT) on spermatogenesis in trans women has already been studied, data on its precise effects on the testicular environment is poor. Therefore, this study aimed to characterize, through histological and transcriptomic analysis, the spermatogonial stem cell niche of 106 trans women who underwent standardized GAHT, comprising estrogens and cyproterone acetate. A partial dedifferentiation of Sertoli cells was observed, marked by the co-expression of androgen receptor and anti-Müllerian hormone which mirrors the situation in peripubertal boys. The Leydig cells also exhibited a distribution analogous to peripubertal tissue, accompanied by a reduced insulin-like factor 3 expression. Although most peritubular myoid cells expressed alpha-smooth muscle actin 2, the expression pattern was disturbed. Besides this, fibrosis was particularly evident in the tubular wall and the lumen was collapsing in most participants. A spermatogenic arrest was also observed in all participants. The transcriptomic profile of transgender tissue confirmed a loss of mature characteristics - a partial rejuvenation - of the spermatogonial stem cell niche and, in addition, detected inflammation processes occurring in the samples. The present study shows that GAHT changes the spermatogonial stem cell niche by partially rejuvenating the somatic cells and inducing fibrotic processes. These findings are important to further understand how estrogens and testosterone suppression affect the testis environment, and in the case of orchidectomized testes as medical waste material, their potential use in research.

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 (TGFβ1)-induced mesenchymal stem cells (MSCs). In this study, we investigated the reproductive efficiency and safety of co-transplanting spermatogonial stem cells (SSCs) and TGFβ1-induced MSCs. Methods A mouse model for long-term infertility was used to transplant SSCs (SSCT, n  = 10) and a combination of SSCs and TGFβ1-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 TGFβ1-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.

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.