Spermatogenesis after gonadotoxic childhood treatment: follow-up of 12 patients.

STUDY QUESTION: What is the long-term impact of presumed gonadotoxic treatment during childhood on the patient's testicular function at adulthood? SUMMARY ANSWER: Although most patients showed low testicular volumes and some degree of reproductive hormone disruption 12.3 (2.3-21.0) years after gonadotoxic childhood therapy, active spermatogenesis was demonstrated in the semen sample of 8 out of the 12 patients. WHAT IS KNOWN ALREADY: In recent decades, experimental testicular tissue banking programmes have been set up to safeguard the future fertility of young boys requiring chemo- and/or radiotherapy with significant gonadotoxicity. Although the risk of azoospermia following such therapies is estimated to be high, only limited long-term data are available on the reproductive potential at adulthood. STUDY DESIGN SIZE DURATION: This single-centre prospective cohort study was conducted between September 2020 and February 2023 and involved 12 adult patients. PARTICIPANTS/MATERIALS SETTING METHODS: This study was carried out in a tertiary care centre and included 12 young adults (18.1-28.3 years old) who had been offered testicular tissue banking prior to gonadotoxic treatment during childhood. All patients had a consultation and physical examination with a fertility specialist, a scrotal ultrasound to measure the testicular volumes and evaluate the testicular parenchyma, a blood test for assessment of reproductive hormones, and a semen analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular tissue was banked prior to the gonadotoxic treatment for 10 out of the 12 included patients. Testicular volumes were low for 9 patients, and 10 patients showed some degree of reproductive hormone disruption. Remarkably, ongoing spermatogenesis was demonstrated in 8 patients at a median 12.3 (range 2.3-21.0) years post-treatment. LIMITATIONS REASONS FOR CAUTION: This study had a limited sample size, making additional research with a larger study population necessary to verify these preliminary findings. WIDER IMPLICATIONS OF THE FINDINGS: These findings highlight the need for multicentric research with a larger study population to establish universal inclusion criteria for immature testicular tissue banking. STUDY FUNDING/COMPETING INTERESTS: This study was conducted with financial support from the Research Programme of the Research Foundation-Flanders (G010918N), Kom Op Tegen Kanker, and Scientific Fund Willy Gepts (WFWG19-03). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: NCT04202094; https://clinicaltrials.gov/ct2/show/NCT04202094?id=NCT04202094&draw=2&rank=1 This study was registered on 6 December 2019, and the first patient was enrolled on 8 September 2020.

A legal framework on advanced practice nursing in Belgium: what do we and don't we know?

OBJECTIVES: Presenting the Belgian new framework for Advanced Practice Nursing (APN) - 'Verpleegkundig Specialist [VS]'/"Infirmier de pratique avancée [IPA]" outlined in the Law of 22 April 2019, followed by a discussion of the lack of clarity, the current challenges and future opportunities. METHODS: The framework was analyzed by an expert in healthcare legislation and discussed by academics in Nursing Science and members of the board of directors of the Belgian Society of APN. RESULTS: Relevant paragraphs within this new law are"Article 46 §1. No one is allowed to carry the title of 'VS/IPA' who does not possess a bachelor in nursing mentioned in article 45 and who does not meet the requirements specified in this article. At the minimum, a master's degree in Nursing Sciences is also required. §2. Additional to the scope of practice of nursing as mentioned in article 46, the 'VS/IPA' perform, in the context of complex nursing care, medical interventions in order to maintain, improve or restore the health of the patient. Care is provided in the context of a specific target group of patients and in close concertation with the physician and potential other healthcare professionals. CONCLUSION: Although the legal recognition of the title of VS/IPA is a major breakthrough that will innovate healthcare, clarification is needed: How do VS/IPA distinguish themselves from other nursing functions, what is complex nursing care, which medical interventions can be performed, what is meant by specific target group of patients, what does 'in close concertation with the physician' entail, and will advisory power be possible?

Toxinotype A Clostridium perfringens causing septicaemia with intravascular haemolysis: two cases and review of the literature.

BACKGROUND: Septicaemia with intravascular haemolysis is a rare, but often fatal, presentation of Clostridium perfringens infection. C. perfringens is a Gram-positive, anaerobic bacterium that can produce multiple toxins. Toxinotyping is not performed regularly. METHODS: This article describes two human cases of C. perfringens infections. Toxinotyping was performed using polymerase chain reaction (PCR). Additionally, a structured review of the literature was performed which searched specifically for cases of C. perfringens infection with haemolysis. RESULTS: Both cases were identified as toxinotype A strains and both cases were fatal. Also, both cases showed marked haemolysis during their clinical course, which is assumed to have played a significant role in their outcome. In total, 83 references were identified describing human C. perfringens infection with haemolysis. Mortality rates have been stable over the last 10 years at 80%. Toxinotyping has been performed in a total of six cases. Of the four cases analysed by PCR, all were identified as toxinotype A. CONCLUSIONS: Haemolytic C. perfringens infections are rare but are fatal in most cases. Toxinotyping is performed rarely. The authors advocate increased use of toxinotyping to gain insight into pathophysiology and more effective interventions.

Public health and clinical approach to proactive management of frailty in multidimensional arena.

Background: Demographic changes have forced communities and people themselves to reshape ageing concepts and approaches and try to develop actions towards active and healthy ageing. In this context, the European Commission launched different private-public partnerships to develop new solutions and answers on questions related to this topic. The European Innovation Partnership on Active and Healthy Ageing, including topic related action groups as well reference sites committed towards a common action to facilitate active and healthy ageing, has contributed key elements for interventions, scaled up best practices and evaluated impact of their action to drive innovation across many regions in Europe over the past years. Methods: This paper describes action taken by A3 action group in the European Innovation Partnership on Active and Healthy Ageing. This paper gives an overview of how the partnership combined the view on frailty coming from public health as well as the clinical management. Results: Within different European regions, to tackle frailty, EIPonAHA partners have conceptualized functional decline and frailty, making use of good practice models working well on community programs. The A3 Group of EIPonAHA has worked alongside a process of innovation, targeting all ageing citizens with the clear goal of involving communities in the preventive approach. Conclusion: Engagement needs of older people with a focus on functionally rather than disease management as primary objective is considered as an overarching concept, also embracing adherence, compliance, empowerment, health literacy, shared decision-making, and activation. Furthermore, training of staff working with ageing people across all sectors needs to be implemented and evaluated in future studies.

Histologic Analysis of Testes from Prepubertal Patients Treated with Chemotherapy Associates Impaired Germ Cell Counts with Cumulative Doses of Cyclophosphamide, Ifosfamide, Cytarabine, and Asparaginase.

Cryopreservation of immature testicular tissue is an experimental strategy for the preservation of fertility in prepubertal boys that will be subjected to a gonadotoxic onset, as is the case of oncologic patients. Therefore, the objective of this study was to assess the impact of chemotherapeutic treatments on the testicular histologic phenotype in prepubertal patients. A total of 56 testicular tissue samples from pediatric patients between 0 and 16 years old (28 with at least one previous chemotherapeutic onset and 28 untreated controls) were histologically analyzed and age-matched compared. At least two 5-µm sections from testis per patient separated by a distance of 100 µm were immunostained for the germ cell marker VASA, the spermatogonial markers UTF1, PLZF, UCHL1, and SALL4, the marker for proliferative cells KI67, and the Sertoli cell marker SOX9. The percentage of tubule cross-sections positive for each marker and the number of positive cells per tubule cross-section were determined and association with the cumulative dose received of each chemotherapeutic drug was statistically assessed. Results indicated that alkylating agents, cyclophosphamide and ifosfamide, but also the antimetabolite cytarabine and asparaginase were associated with a decreased percentage of positive tubules and a lower number of positive cells per tubule for the analyzed markers. Our results provide new evidences of the potential of chemotherapeutic agents previously considered to have low gonadotoxic effects such as cytarabine and asparaginase to trigger a severe testicular phenotype, hampering the potential success of future fertility restoration in experimental programs of fertility preservation in prepubertal boys.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model.

STUDY QUESTION: Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip? SUMMARY ANSWER: Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1 week and the co-cultures reproduced specific natural and drug-induced liver-testis systemic interactions. WHAT IS KNOWN ALREADY: Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver-testis axis. However, these are important to study the biotransformation of substances. STUDY DESIGN, SIZE, DURATION: Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver-testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated. PARTICIPANTS/MATERIALS, SETTING, METHODS: The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured. WIDER IMPLICATIONS OF THE FINDINGS: This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

Spotlight on avian pathology: untangling contradictory disease descriptions of necrotic enteritis and necro-haemorrhagic enteritis in broilers.

Necrotic enteritis (NE) is one of the most detrimental infectious diseases in the modern poultry industry, characterized by necrosis in the small intestine. It is commonly accepted that NetB-producing C. perfringens type G strains are responsible for the disease. However, based on both macroscopic and histopathological observations, two distinct types of NE are observed. To date, both a haemorrhagic form of NE and the type G-associated non-haemorrhagic disease entity are commonly referred to as NE and the results from scientific research are interchangeably used, without distinguishing between the disease entities. Therefore, we propose to rename the haemorrhagic disease entity to necro-haemorrhagic enteritis.

From the bench to practice - Field integration of community-based services for older citizens with different levels of functional limitation across European Regions.

The meeting of the European Innovation Partnership on Active and Healthy Ageing (EIPonAHA) action group A3 together with members of the Reference site collaborative network (RSCN) in December 2019 in Rome focused on integration of evidence-based approaches on health and care delivery for older citizens at different levels of needs with expertise coming from stakeholder across Europe. It was the final aim of the group to co-create culturally sensitive pathways and facilitate co-ownership for further implementation of the pathways in different care systems across Europe. The study design is a mixed method approach. Based on data analysis from a cohort of community-dwelling over-65 citizens in the framework of a longitudinal observational study in Rome, which included health, social and functional capacity data, three personas profiles were developed: the pre-frail, the frail and the very frail personas. Based on these data, experts were asked to co-create care pathways due to evidence and eminence during a workshop and included into a final report. All working groups agreed on a common understanding that integration of care means person-centered integration of health and social care, longitudinally provided across primary and secondary health care including citizens' individual social, economic and human resources. Elements for consideration during care for pre-frail people are loneliness and social isolation, which, lead to limitation of physical autonomy in the light of reduced access to social support. Frail people need adaption of environmental structures and, again, social resource allocation to maintain at home. Very frail are generally vulnerable patients with complex needs. Most of them remain at home because of a strong individual social support and integrated health care delivery. The approach described in this publication may represent a first approach to scaling-up care delivery in a person-centered approach.

Health tourism: an opportunity for sustainable development.

In February 2017, the "Programma Mattone Internazionale Salute" (ProMis), that is the Italian Program for Internationalization of Regional Health Systems of the Ministry of Health (MoH), presented the first version of its Position Paper on Health Tourism, which embeds a first shared approach to the recommendations expressed by the European Committee of Regions (CoR) on "Age-Friendly" tourism. The CoR stresses the importance of local and regional authorities in the coordination of multi-sectoral policies such as healthcare, social assistance, transport, urban planning and rural development in relation to the promotion of mobility, security, accessibility of services, including health care and social services. "Age-friendly" tourism is an example of an innovative tourist offer that strives to meet the health needs of the entire "traveling" population, with an integrated and cross-sector approach that involves various organizations operating in sectors such as healthcare, accessibility and transport. The aim of the workshop was to explore the interest of the stakeholders to participate in a systemic action in the field of "health" tourism, and to identify priority implementation areas that offer opportunities to take advantage of validated, innovative experiences that strengthen the accessibility to health and social services in regional, national and international contexts. This effort provides the opportunity to take advantage of aligning the European Structural and Investment Funds (ESIF) to the development of tourism, coherently with the needs and resources of local and regional health authorities.

Rapid growth predisposes broilers to necrotic enteritis.

Over the past 50 years, intentional genetic selection within the broiler industry has led to major improvements in both body weight gain (BWG) and feed conversion efficiency. Next to its economic advantages, enhancing BWG can increase the risk of metabolic and skeletal disorders. The aim of this study was to examine whether higher BWG is a predisposing factor for broiler necrotic enteritis. In this study, 300 broilers were challenged with Clostridium perfringens using a well-established, previously described challenge model. It was found that birds with higher body weight (BW) and BWG before challenge were predisposed to develop more severe necrotic enteritis lesions. After challenge, the average BWG of the birds developing mild to severe lesions dropped significantly, negatively affecting bird welfare and performance. These results show a significant interplay between BWG and the development of necrotic enteritis lesions. This raises the question whether there is a limit to broiler performance with respect to maintaining intestinal health, and whether decreasing BWG (at certain stages of the growth cycle) can be part of a plan to prevent intestinal pathology. RESEARCH HIGHLIGHTS Higher body weight is a predisposing factor to necrotic enteritis in broilers.

Complete spermatogenesis in intratesticular testis tissue xenotransplants from immature non-human primate.

STUDY QUESTION: Can full spermatogenesis be achieved after xenotransplantation of prepubertal primate testis tissue to the mouse, in testis or subcutaneously? SUMMARY ANSWER: Intratesticular xenotransplantation supported the differentiation of immature germ cells from marmoset (Callithrix jacchus) into spermatids and spermatozoa at 4 and 9 months post-transplantation, while in subcutaneous transplants, spermatogenic arrest was observed at 4 months and none of the transplants survived at 9 months. WHAT IS KNOWN ALREADY: Auto-transplantation of cryopreserved immature testis tissue (ITT) could be a potential fertility restoration strategy for patients with complete loss of germ cells due to chemo- and/or radiotherapy at a young age. Before ITT transplantation can be used for clinical application, it is a prerequisite to demonstrate the feasibility of the technique and identify the conditions required for establishing spermatogenesis in primate ITT transplants. Although xenotransplantation of ITT from several species has resulted in complete spermatogenesis, in human and marmoset, ITT has not been successful. STUDY DESIGN, SIZE, DURATION: In this study, we used marmoset as a pre-clinical animal model. ITT was obtained from two 6-month-old co-twin marmosets. A total of 147 testis tissue pieces (~0.8-1.0 mm3 each) were transplanted into the testicular parenchyma (intratesticular; n = 40) or under the dorsal skin (ectopic; n = 107) of 4-week-old immunodeficient Swiss Nu/Nu mice (n = 20). Each mouse received one single marmoset testis tissue piece in each testis and 4-6 pieces subcutaneously. Xenotransplants were retrieved at 4 and 9 months post-transplantation and evaluations were performed with regards to transplant survival, spermatogonial quantity and germ cell differentiation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Transplant survival was histologically evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. Spermatogonia were identified by MAGE-A4 via immunohistochemistry. Germ cell differentiation was assessed by morphological identification of different germ cell types on H/PAS stained sections. Meiotically active germ cells were identified by BOLL expression. CREM immunohistochemistry was performed to confirm the presence of post-meiotic germ cells and ACROSIN was used to determine the presence of round, elongating and elongated spermatids. MAIN RESULTS AND THE ROLE OF CHANCE: Four months post-transplantation, 50% of the intratesticular transplants and 21% of the ectopic transplants were recovered (P = 0.019). The number of spermatogonia per tubule did not show any variation. In 33% of the recovered intratesticular transplants, complete spermatogenesis was established. Overall, 78% of the intratesticular transplants showed post-meiotic differentiation (round spermatids, elongating/elongated spermatids and spermatozoa). However, during the same period, spermatocytes (early meiotic germ cells) were the most advanced germ cell type present in the ectopic transplants. Nine months post-transplantation, 50% of the intratesticular transplants survived, whilst none of the ectopic transplants was recovered (P < 0.0001). Transplants contained more spermatogonia per tubule (P = 0.018) than at 4 months. Complete spermatogenesis was observed in all recovered transplants (100%), indicating a progressive spermatogenic development in intratesticular transplants between the two time-points. Nine months post-transplantation, transplants contained more seminiferous tubules with post-meiotic germ cells (37 vs. 5%; P < 0.001) and fewer tubules without germ cells (2 vs. 8%; P = 0.014) compared to 4 months post-transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although xenotransplantation of marmoset ITT was successful, it does not fully reflect all aspects of a future clinical setting. Furthermore, due to ethical restrictions, we were not able to prove the functionality of the spermatozoa produced in the marmoset transplants. WIDER IMPLICATIONS OF THE FINDINGS: In this pre-clinical study, we demonstrated that testicular parenchyma provides the required microenvironment for germ cell differentiation and long-term survival of immature marmoset testis tissue, likely due to the favourable temperature regulation, growth factors and hormonal support. These results encourage the design of new experiments on human ITT xenotransplantation and show that intratesticular transplantation is likely to be superior to ectopic transplantation for fertility restoration following gonadotoxic treatment in childhood. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by the ITN Marie Curie Programme 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568) and the scientific Fund Willy Gepts from the UZ Brussel (ADSI677). D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome?

STUDY QUESTION: When does germ cell loss and fibrosis occur in patients with Klinefelter syndrome (KS)? SUMMARY ANSWER: In KS, germ cell loss is not observed in testicular tissue from fetuses in the second semester of pregnancy but present at a prepubertal age when the testicular architecture is still normal, while fibrosis is highly present at an adolescent age. WHAT IS KNOWN ALREADY: Most KS patients are azoospermic at adult age because of a massive germ cell loss. However, the timing when this germ cell loss starts is not known. It is assumed that germ cell loss increases at puberty. Therefore, testicular sperm extraction (TESE) at an adolescent age has been suggested to increase the chances of sperm retrieval at onset of spermatogenesis. However, recent data indicate that testicular biopsies from peripubertal KS patients contain only a few germ cells. STUDY DESIGN, SIZE, DURATION: In this study, we give an update on fertility preservation in adolescent KS patients and evaluate whether fertility preservation would be beneficial at prepubertal age. The possibility of retrieving testicular spermatozoa by TESE was evaluated in adolescent and adult KS men. The presence of spermatogonia and the degree of fibrosis were also analysed in testicular biopsies from KS patients at different ages. The patients were divided into four age groups: foetal (n = 5), prepubertal (aged 4-7 years; n = 4), peripubertal (aged 12-16 years; n = 20) and adult (aged 18-41 years; n = 27) KS patients. PARTICIPANTS/MATERIALS, SETTING, METHODS: In peripubertal and adult KS patients, retrieval of spermatozoa was attempted by semen analysis after masturbation, vibrostimulation, electroejaculation or by TESE. MAGE-A4 immunohistochemistry was performed to evaluate the presence of germ cells in testicular biopsies from foetal, prepubertal, peripubertal and adult KS patients. Tissue morphology was evaluated by haematoxylin-periodic acid Schiff (H/PAS) staining. MAIN RESULTS AND THE ROLE OF CHANCE: Testicular spermatozoa were collected by TESE in 48.1% of the adult KS patients, while spermatozoa were recovered after TESE in only one peripubertal patient (5.0%). Germ cells were detectable in testicular biopsies from 21% of adult men for whom no spermatozoa could be retrieved by TESE and in 31.5% of peripubertal KS boys. Very small numbers of spermatogonia (0.03-0.06 spermatogonia/tubule) were detected in three out of four (75%) prepubertal patients. At a foetal age, the number of germ cells was similar for KS and control samples. Increased signs of fibrosis were not present at foetal and prepubertal ages, but peripubertal and adult KS patients showed high levels of fibrosis. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Only four prepubertal biopsies were included in this study, but they all showed a very low germ cell number. A high variability in the number of spermatogonia per mm2 was observed in the limited (n = 5) number of foetal biopsies. However, testicular biopsies from prepubertal and foetal Klinefelter patients are difficult to obtain. WIDER IMPLICATIONS OF THE FINDINGS: Testicular tissue banking at a prepubertal age has been suggested as a potential method for fertility preservation in early diagnosed KS boys. However, our results show that a reduction in germ cell number has already taken place in childhood. Therefore, offering testicular tissue banking in young KS boys to prevent subsequent sterility might be a questionable strategy. However, this should be confirmed in a larger study population. STUDY FUNDING/COMPETING INTEREST(S): This project was funded by the scientific Fund Willy Gepts from the UZ Brussel (D.V.S., J.D.S.), grants from the Vrije Universiteit Brussel (E.G.) and a Methusalem grant (K.S.). D.V.S is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2815N). No conflict of interest is declared.

Human in vitro spermatogenesis from pluripotent stem cells: in need of a stepwise differentiation protocol?

Recently, complete in vitro generation of male gametes starting from pluripotent stem cells was obtained in a mouse model with live offspring as a result. This breakthrough was probably due to the use of a stepwise differentiation protocol taking the tightly regulated in vivo situation into account. As shown previously, factors of the TGFß superfamily, metabolites of vitamin A, growth hormones, sex steroids and, most importantly, somatic cell support are major regulators of the development, survival, proliferation and differentiation of male gametes. However, up till now, all differentiation protocols starting from human pluripotent stem cells only focused on one or two of these substantive factors, not taking any timeframe into account, leading to promising but unsatisfying results with low efficiency. Therefore, progress might be achieved by including a stepwise differentiation protocol, including all proven contributing regulators, and therefore mimicking more closely human in vivo spermatogenesis and its temporo-spatial organization. In this review, the indispensable regulators of in vivo spermatogenesis and the outcomes of related human in vitro studies are discussed with the aim of unravelling the most successful combinations of medium factors to be used in future differentiation protocols.

Attitudes of nurses towards family involvement in the care for patients with cardiovascular diseases.

INTRODUCTION: There is increasing evidence supporting the relationship between family support and patient outcomes. Therefore, involving families in the care of cardiovascular patients is expected to be beneficial for patients. The quality of the encounter with families highly depends on the attitudes of nurses towards the importance of families in patient care. AIM: The aim of this study was to describe the attitudes of nurses towards family involvement in patient care and to investigate the individual contributions of demographic, professional and regional background characteristics. METHOD: A survey was distributed among cardiovascular nurses attending an international conference in Norway and a national conference in Belgium. Nurses were asked to complete a questionnaire, including the Families' Importance in Nursing Care - Nurses' Attitudes scale. The study population consisted of respondents from Belgium ( n = 348) and from Scandinavian countries (Norway, Sweden and Denmark; n = 77). RESULTS: In general, nurses viewed the family as important in care. However, attitudes towards actively inviting families to take part in patient care were less positive. Higher educational level and a main practice role in research, education or management were significantly associated with more positive attitudes. Furthermore, the attitudes of respondents living in Scandinavia were more positive as compared to the attitudes of respondents living in Belgium. CONCLUSION: Education on the importance of families and active family involvement in patient care seems to be necessary in basic, undergraduate education, but also in clinical practice. More research is necessary in order to explore the cultural and regional differences in the attitudes of nurses towards the involvement of families in patient care.

Can mesenchymal stem cells improve spermatogonial stem cell transplantation efficiency?

Improved treatments have led to an increased survival rate in cancer patients. However, in pre-pubertal boys, these gonadotoxic treatments can result in the depletion of the spermatogonial stem cell (SSC) pool causing lifelong infertility. SSC transplantation has been proposed as a promising technique to preserve the fertility of these patients. In mice, this technique has resulted in live-born offspring, but the efficiency of colonization remained low. This could be because of a deficient microenvironment, leading to apoptosis of the transplanted SSCs. Interestingly, mesenchymal stem cells (MSCs), being multipotent and easy to isolate and multiply in vitro, are nowadays successfully and widely used in regenerative medicine. Here, we shortly review the current understanding of MSC and SSC biology, and we hypothesize that a combined MSC-SSC transplantation might improve the efficiency of SSC colonization and differentiation as paracrine factors from MSCs may contribute to the SSC niche.

Non-toxic perfringolysin O and α-toxin derivatives as potential vaccine candidates against bovine necrohaemorrhagic enteritis.

Bovine necrohaemorrhagic enteritis is a fatal Clostridium perfringens type A-induced disease that is characterised by sudden death. Recently the involvement of perfringolysin O and α-toxin in the development of necrohaemorrhagic lesions in the gut of calves was suggested, and thus derivatives of these toxins are potentially suitable as vaccine antigens. In the current study, the perfringolysin O derivative PFOL491D, alone or in combination with α-toxin derivative GST-cpa247-370, was evaluated as possible vaccine candidate, using in vitro assays. PFOL491D showed no haemolytic effect on horse red blood cells and no cytotoxic effect on bovine endothelial cells. Furthermore, calves immunised with PFOL491D raised antibodies against perfringolysin O that could inhibit the perfringolysin O-associated haemolytic activity on horse red blood cells. Antisera from calves immunised with PFOL491D had a significantly higher neutralising capacity against the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells than serum from control calves (P <0.05). Immunisation of calves with PFOL491D in combination with GST-cpa247-370 elicited antibodies against perfringolysin O and α-toxin and consequently inhibited both the perfringolysin O-associated haemolytic activity and the α-toxin-associated lecithinase activity in vitro. Additionally, the neutralising ability of these antisera on the cytotoxic effect of C. perfringens culture supernatant to bovine endothelial cells was significantly higher than that from calves immunised with PFOL491D (P <0.001). In conclusion, perfringolysin O derivative PFOL491D is an immunogenic antigen that can potentially be used to produce vaccine against bovine necrohaemorrhagic enteritis. Including α-toxin derivative GST-cpa247-370 has an additional protective effect and therefore vaccination of calves with a combination of both antigens seems even more promising.

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation.

BACKGROUND: Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE: This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS: An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES: The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. WIDER IMPLICATIONS: Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration.

Development of a Quality of Meals and Meal Service Set of Indicators for Residential Facilities for Elderly.

OBJECTIVES: To develop a content validated set of indicators to evaluate the quality of meals and meal service in residential facilities for elderly. Inadequate food intake is an important risk factor for malnutrition in residential facilities for elderly. Through better meeting the needs and preferences of residents and optimization of meals and meal service, residents' food intake can improve. No indicators were available which could help to guide strategies to improve the quality of meals and meal service. DESIGN: The indicator set was developed according to the Indicator Development Manual of the Dutch Institute for Health Care Improvement (CBO). The working group consisted of three nurse researchers and one expert in gastrology and had expertise in elderly care, malnutrition, indicator development, and food quality. A preliminary list of potential indicators was compiled using the literature and the working group's expertise. Criteria necessary to measure the indicator in practice were developed for each potential indicator. In a double Delphi procedure, the list of potential indicators and respective criteria were analyzed for content validity, using a multidisciplinary expert panel of 11 experts in elderly meal care. RESULTS: A preliminary list of 20 quality indicators, including 45 criteria, was submitted to the expert panel in a double Delphi procedure. After the second Delphi round, 13 indicators and 25 criteria were accepted as having content validity. The content validity index (CVI) ranged from 0.83 to 1. The indicator set consisted of six structural, four result, and three outcome indicators covering the quality domains food, service and choice, as well as nutritional screening. The criteria measure diverse aspects of meal care which are part of the responsibility of kitchen staff and health care professionals. CONCLUSION: The 'quality of meals and meal service' set of indicators is a resource to map meal quality in residential facilities for elderly. As soon as feasibility tests in practice are completed, the indicator set can be used to guide meal and meal service quality improvement projects in collaboration with kitchen staff and health care professionals. These improvement projects will help to improve food intake and reduce the risk of malnutrition among elders living in residential facilities.

The multifactorial nature of microRNAs in vascular remodelling.

Vascular remodelling is a multifactorial process that involves both adaptive and maladaptive changes of the vessel wall through, among others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial, e.g. during neovascularization after ischaemia, as well as pathological, e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs. A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease (CVD). The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various CVDs.