Fonds National De La Recherche Research Articles

Influence of ovarian stromal cells on human ovarian follicle growth in a 3D environment.

Do ovarian stromal cells (OSCs) influence the viability and growth of human preantral follicles in vitro? A feeder layer of OSCs promotes the growth and transition of low developmental stage follicles to the primary/secondary stage while maintaining a high proportion of viable follicles. In the ovary, follicles rely on the support of ovarian cells, which secrete essential factors for their survival and development. This phenomenon has also been demonstrated in vitro through the 3D culture of isolated mouse primary and secondary follicles on a feeder layer of OSCs. This co-culture notably enhances follicle survival and growth. Pre-antral follicles were isolated from human frozen-thawed ovarian tissue biopsies and then encapsulated in 1% alginate scaffolds. These embedded preantral follicles were either placed directly on the OSCs feeder layer or at the bottom of a culture dish for a 7-day in vitro culture (control). The study compared follicle viability, growth, and hormone production between the different groups. Primordial/intermediate and primary follicles were isolated from frozen-thawed ovarian tissue of cancer patients (n = 6). OSCs were then isolated from ovarian tissue of post-menopausal women and cultured as a feeder layer. Follicle diameter was measured on Days 0 and 7 using an inverted microscope to assess their development based on the increase in diameter. Viability was evaluated by staining a subset of follicles (n = 87) with calcein AM and ethidium homodimer-I, followed by classification into healthy/minimally damaged and damaged/dead follicles using confocal fluorescence microscopy. Additionally, estradiol levels were measured using ELISA. A total of 382 human preantral follicles (370 primordial/intermediate and 12 primary) with a mean diameter of 40.8 ± 9.9 µm (mean ± SD) were isolated, embedded in 1% alginate hydrogel, and placed either on a monolayer of OSCs or directly on the plastic. By Day 7, the preantral follicles showed a significant size increase under both culture conditions (P < 0.0001 for D0 vs D7). The mean diameter of follicles (quiescent and growing) cultured on the feeder layer was 80.6 ± 11.0 μm compared to 67.3 ± 7.2 μm without it (P = 0.07). During the 7-day in vitro culture, the viability of the follicles significantly decreased only in the group without an OSCs monolayer compared to the D0 viability (P < 0.05). Additionally, more follicles transitioned to a higher developmental stage in the presence of OSCs (D0 primordial/intermediate: 184, primary: 7 vs D7 primordial/intermediate: 51, primary/secondary: 93) compared to those cultured without OSCs (D0 primordial/intermediate: 186, primary: 5 vs D7 primordial/intermediate: 84, primary/secondary: 65; P < 0.001). Specifically, 66 and 44 follicles reached the secondary stage (75< x <200 μm) in the presence and absence of OSCs, respectively. Moreover, the estradiol level was significantly higher (P = 0.006) in the alginate beads containing primordial and growing follicles cultured on the OSCs (54.1 ± 14.2 pg/ml) compared to those cultured without OSCs (29.9 ± 4.0 pg/ml). N/A. This study was conducted using a short-term culture, and none of the primordial/intermediate/primary follicles reached the antral stage. Further in vitro studies are required to investigate follicular developmental capacity, physiology, and steroidogenesis in alginate scaffolds with human OSCs. Activating and growing human primordial/intermediate follicles to a secondary stage in in vitro short-term culture has posed a longstanding challenge. However, co-culturing with human OSCs has shown the potential to overcome this limitation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR Convention grant number T.0004.20 awarded to C.A.A., PhD scholarship awarded to H.V.), Fondation Louvain (awarded to C.A.A.; PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer), Foundation Against Cancer (grant 2018-042 awarded to A.C.), and the European Community Structural Funds and Lithuanian Research Council (Agreement registration No. D-19-0874). The authors have no conflicts of interest to declare.

Locus coeruleus activity while awake is associated with REM sleep quality in older individuals.

The locus coeruleus (LC) is the primary source of norepinephrine in the brain and regulates arousal and sleep. Animal research shows that it plays important roles in the transition between sleep and wakefulness, and between slow wave sleep and rapid eye movement sleep (REMS). It is unclear, however, whether the activity of the LC predicts sleep variability in humans. We used 7 Tesla functional Magnetic Resonance Imaging, sleep electroencephalography (EEG) and a sleep questionnaire to test whether the LC activity during wakefulness was associated with sleep quality in 33 healthy younger (~22y; 28 women) and 19 older (~61y; 14 women) individuals. We found that, in older, but not in younger participants, higher LC activity, as probed during an auditory attentional task, was associated with worse subjective sleep quality and with lower power over the EEG theta band during REMS. The results remained robust even when accounting for the age-related changes in the integrity of the LC. These findings suggest that LC activity correlates with the perception of the sleep quality and an essential oscillatory mode of REMS, and that the LC may be an important target in the treatment of sleep and age-related diseases. This work was supported by Fonds National de la Recherche Scientifique (FRS-FNRS, T.0242.19 & J. 0222.20). Action de Recherche Concertée - Fédération Wallonie-Bruxelles (ARC SLEEPDEM 17/27-09), Fondation Recherche Alzheimer (SAO-FRA 2019/0025), University of Liège, European Regional Development Fund (Radiomed & Biomed-Hub).

Use of mesenchymal stem cells to enhance or restore fertility potential: a systematic review of available experimental strategies.

To what extent does regenerative medicine with stem cell therapy help to address infertility issues for future clinical application? Regenerative medicine using different stem cell sources is yielding promising results in terms of protecting the ovarian reserve from damage and senescence, and improving fertility potential in various preclinical settings. Regenerative medicine using stem cell therapy is emerging as a potential strategy to address a number of issues in the field of human reproduction. Indeed, different types of adult and fetal mesenchymal stem cells (MSCs) have been tested with promising results, owing to their ability to differentiate into different tissue lineages, move toward specific injured sites (homing), and generate a secretome with wound-healing, proangiogenic, and antioxidant capacities. Guided by the checklist for preferred reporting items for systematic reviews and meta-analyses, we retrieved relevant studies from PubMed, Medline, and Embase databases until June 2023 using the following keywords: 'mesenchymal stem cells' AND 'ovarian follicles' OR 'ovarian tissue culture' OR 'ovarian follicle culture' OR 'cumulus oocyte complex'. Only peer-reviewed published articles written in English were included. The primary outcome for the experimental strategies was evaluation of the ovarian reserve, with a focus on follicle survival, number, and growth. Secondary outcomes involved analyses of other parameters associated with the follicle pool, such as hormones and growth factors, ovarian tissue viability markers including oxidative stress levels, oocyte growth and maturation rates, and of course pregnancy outcomes. Preclinical studies exploring MSCs from different animal origins and tissue sources in specific conditions were selected (n = 112), including: in vitro culture of granulosa cells, ovarian tissue and isolated ovarian follicles; ovarian tissue transplantation; and systemic or intraovarian injection after gonadotoxic or age-related follicle pool decline. Protecting the ovarian reserve from aging and gonadotoxic damage has been widely tested in vitro and in vivo using murine models and is now yielding initial data in the first ever case series of patients with premature ovarian insufficiency. Use of MSCs as feeder cells in ovarian tissue culture was found to improve follicle outcomes and oocyte competence, bringing us one step closer to future clinical application. MSCs also have proved effective at boosting revascularization in the transplantation site when grafting ovarian tissue in experimental animal models. While preclinical results look promising in terms of protecting the ovarian reserve in different experimental models (especially those in vitro using various mammal experimental models and in vivo using murine models), there is still a lot of work to do before this approach can be considered safe and successfully implemented in a clinical setting. All gathered data on the one hand show that regenerative medicine techniques are quickly gaining ground among innovative techniques being developed for future clinical application in the field of reproductive medicine. After proving MSC effectiveness in preclinical settings, there is still a lot of work to do before MSCs can be safely and effectively used in different clinical applications. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0077.14, FNRS-CDR J.0063.20, and grant 5/4/150/5 awarded to Marie-Madeleine Dolmans), Fonds Spéciaux de Recherche, and the Fondation St Luc. None of the authors have any competing interest to disclose. N/A.

Importance of oxygen tension in human ovarian tissue in vitro culture.

Is there any difference between 20% and 5% oxygen (O2) tension in vitro culture (IVC) on the viability and quality of human follicles contained in cultured ovarian cortex? An O2 tension of 5% yields higher follicle viability and quality than does 20% O2 tension after 6 days of IVC. The primordial follicle (PMF) pool resides within the ovarian cortex, where the in vivo O2 tension ranges between 2% and 8%. Some studies suggest that lowering O2 tension to physiological levels may improve in vitro follicle quality rates. This prospective experimental study included frozen-thawed ovarian cortex from six adult patients (mean age: 28.5 years; age range: 26-31 years) who were undergoing laparoscopic surgery for non-ovarian diseases. Ovarian cortical fragments were cultured for 6 days at (i) 20% O2 with 5% CO2 and (ii) 5% O2 with 5% CO2. Non-cultured fragments served as controls. Cortical fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; Ki67 staining to evaluate PMF proliferation; cleaved caspase-3 immunostaining to identify follicle apoptosis; 8-hydroxy-2-deoxyguanosine and gamma-H2AX (γH2AX) immunolabeling to detect oxidative stress damage and DNA double-strand breaks (DSBs) in oocytes and granulosa cells (GCs); and β-galactosidase staining to assess follicle senescence. Droplet digital PCR was also performed to further explore the gene expression of superoxide dismutase 2 (SOD2) and glutathione peroxidase 4 (GPX4) from the antioxidant defense system and cyclin-dependent kinase inhibitors (p21 and p16) as tissue senescence-related genes. Apoptosis (P = 0.002) and follicle senescence (P < 0.001) rates were significantly lower in the 5% O2 group than in the 20% O2 group. Moreover, GCs in follicles in the 20% O2 group exhibited significantly (P < 0.001) higher oxidative stress damage rates than those in the 5% O2 group. DNA DSB damage rates in GCs of follicles were also significantly higher (P = 0.001) in the 20% O2 group than in the 5% O2 group. SOD2 expression was significantly greater in the 5% O2 group compared to the 20% O2 group (P = 0.04) and the non-cultured group (P = 0.002). Expression of p21 was significantly increased in both the 20% O2 (P = 0.03) and 5% O2 (P = 0.008) groups compared to the non-cultured group. Moreover, the 20% O2 group showed significantly greater p16 expression (P = 0.04) than the non-cultured group, while no significant variation was observed between the 5% O2 and no culture groups. N/A. This study focuses on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue. The impact of O2 tension in further steps, such as secondary follicle isolation and maturation, was not investigated here. Our findings suggest that 5% O2 tension culture is a promising step toward potentially solving the problem of poor follicle viability after IVC. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS-PDR T.0064.22, CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.). The authors have nothing to disclose.

Mental health and homelessness in the social service providers' outlook (Luxembourg case)

Purpose The purpose of this paper is to explore the perception of the mental health problems of the homeless population in a high-income country (Luxembourg) by social service providers and to develop proposals for better inclusion of homeless people into the mental health services and homeless people with mental health issues into society. Design/methodology/approach The study was of qualitative design and conducted using a semi-structured interview method (in person). The semi-structured interviews (seven participants) were conducted to analyse the challenges, practice approaches and prospects of stakeholders or decision-makers working in housing exclusion and homelessness. A secondary thematic analysis of this content regarding mental health issues was performed. Findings Three main themes in the social providers’ perception were identified related to mental health and homelessness: the general view on the mental health problems of homeless people (accent on substance use disorders [SUDs], overshadowing of other mental health conditions by the SUDs); the positive impact of housing and social services on the mental health of the homeless per se (role of social rhythms, social connectedness and multidisciplinary approach are emphasised); and the need for improvement of mental health services in the country (need for the long-term timely continuing mental health support and recognition of the importance of complex intersectional and multidisciplinary solutions). Research limitations/implications Mental health themes were not the primary focus while research was planned and conducted. They were revealed as results of secondary qualitative data analysis. Therefore, additional mental health-focused mixed methods research is needed to verify the conclusions. The paper is written on the results of the research project “Social Housing and Homelessness” (SOHOME), implemented at the University of Luxembourg with the financial support of the Fonds National de la Recherche of Luxembourg (FNR12626464). The sponsor had no involvement in the study design, the collection, analysis and interpretation of data or the preparation of the paper. Practical implications The study brings together different perspectives from social workers, stakeholders and decision-makers. The results show that there are cross-field connections between homelessness and mental health that require specialised and coordinated services. The first existing approaches seem to be promising in their continuation but need to be promoted by social policy. Social implications To promote social cohesion in the Luxembourgish society and also to include one of the most vulnerable people, the study points to the importance of the link between homelessness and compromised mental health. Appropriate support and service provision as well as social and affordable housing play a central role. Originality/value To the best of the authors’ knowledge, this study is the first of its kind, revealing several social work stakeholders’ perspective on the mental health of homeless people in Luxembourg.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. N/A.

Editorial introductions.

Editorial introductions.

Role of apoptosis and autophagy in ovarian follicle pool decline in children and women diagnosed with benign or malignant extra-ovarian conditions.

Which biological mechanisms are responsible for physiological ovarian reserve decline owing to aging, or pathological follicle depletion triggered by inflammation or a pro-oxidant environment throughout a woman's lifetime? Ovarian follicle pool size is modulated by both apoptosis and autophagy, the first responsible for its physiological decline over time and increasing in the event of prior chemotherapy in children, and the latter playing a major role in physiological ovarian follicle pool diminution before puberty. Among the different pathways of controlled cell death, apoptosis and autophagy are implicated in follicle loss. Apoptosis participates in eliminating damaged follicles, such as those impaired by chemotherapy (CHT), but its involvement in physiological age-related follicle decline is less well understood. Autophagy has proved crucial in follicle quiescence maintenance in murine models, but its contribution to human follicle pool modulation is still unclear. This retrospective study included 84 patients with benign or malignant extra-ovarian conditions aged between 1 and 35 years, with ovarian tissue stored for histological analyses at the time of cryopreservation (between 2012 and 2021) at a tertiary care center. Ovarian fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; cleaved caspase-3 immunostaining to identify follicle apoptosis; and microtubule-associated proteins 1A/1B light chain 3B immunolabeling to detect follicle autophagy. Transmission electron microscopy was also carried out to investigate ultrastructural features of oocytes and granulosa cells. All analyses stratified patients by age, menarchal status (premenarchal = 32; postmenarchal = 52), potentially gonadotoxic CHT before cryopreservation (n = 14), presence of endometriosis and use of hormonal treatment. Premenarchal patients had a larger follicle pool in terms of total follicle density [mean, range 4979.98 (342.2-21789) versus 918.8 (26.18-3983), P < 0.001], but higher rates of morphologically abnormal [8.52 (0-25.37)% versus 3.54 (0-17.5)%, P < 0.001] and atretic [15.8 (0‒31.85)% versus 10.6 (0-33.33)%, P < 0.01] follicles than postmenarchal subjects. Apoptosis rates did not change with increasing age [27.94 (0-93.2)% in prepubertal subjects and 29.5 (0-100)% in postpubertal subjects], but autophagic follicles were around 10 times more common in premenarchal than postmenarchal subjects [10.21 (0-62.3)% versus 1.34 (0-25)%, P < 0.001], playing a crucial role in age-related follicle decline and elimination of 'abnormal' follicles, that are rarely seen after menarche. The impact of diagnosis and previous CHT varied according to age. In premenarchal patients with previous CHT, significantly more apoptotic [40.22 (0-100)% versus 26.79 (0-87)%, P < 0.05] and fewer abnormal [3.84 (0-10-76)% versus 9.83 (0-25.37)%, P < 0.01] follicles were detected than in subjects with no CHT prior to ovarian tissue cryopreservation, suggesting a direct effect on follicle elimination, especially of those with abnormalities. In postmenarchal subjects with previous CHT, quiescent follicle rates were lower than in patients with no CHT before tissue freezing [71.57 (0-100)% versus 85.89 (50-100)%, P < 0.05], suggesting accelerated follicle activation and growth. Moreover, increased autophagic activity was observed in the event of a cancer diagnosis compared to benign conditions after puberty [26.27 (0-100)% versus 9.48 (0-29.41)%, respectively, P < 0.05]. The impact of specific CHT protocols could not be investigated since the group of patients with previous CHT was highly heterogeneous. This study yields a deeper understanding of regulation of the follicle pool decline, showing for the first time that both apoptosis and autophagy pathways are involved in physiological follicle depletion, the latter being crucial before puberty. Moreover, our data showed a different response to non-physiological damage according to age, with higher apoptosis rates only in premenarchal subjects with previous CHT, confirming that this pathway is activated by drugs known to induce DNA damage in oocytes, such as alkylating agents, but not by cancer itself. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (F.R.S.-FNRS/FRIA FC29657 awarded to L.C., CDR J.0063.20 and grant 5/4/150/5 awarded to M.M.D.), grants from the Fondation contre le Cancer (grant 2018-042 awarded to A.Ca.), the Fondazione Comunitaria del Varesotto and Provincia di Varese ('Amalia Griffini' Fellowship in Gynecology and Obstetrics awarded to A.Ce.), Fonds Spéciaux de Recherche, Fondation St Luc and donations from the Ferrero family. The authors have no competing interests to declare. N/A.

Prognostic implications of paradoxical low gradient severe aortic stenosis: a comprehensive analysis from a large multicentric registry

Abstract Background Up to 40% of patients with severe aortic stenosis (SAS; indexed aortic valve area (AVAi) &amp;lt;0.6 cm2/m2) present with low transvalvular mean gradient (MG) despite a normal left ventricular ejection fraction (EF). There is intense debate about the prognostic significance of such entity, with some referring to it as an advanced form of the disease, others as an intermediate form between a moderate and a severe form. Objectives To compare outcome of patients with paradoxical low gradient SAS (PLG-SAS; i.e., mean gradient &amp;lt;40 mmHg and AVAi &amp;lt;0.6 cm2/m2) vs. moderate aortic stenosis (MAS; i.e. mean gradient &amp;lt;40 mmHg and AVAi &amp;gt;0.6 cm2/m2) and high gradient SAS (HG-SAS; i.e. mean gradient &amp;gt;40 mmHg and AVAi &amp;lt;0.6 cm2/m2). Methods 2582 consecutive patients with aortic stenosis (PLG-SAS, n=933; MAS, n=876 and HG-SAS, n=773) and a preserved EF (&amp;gt;50%) from an international multicentric registry were studied. Five years mortality between groups was compared using Kaplan Meier analysis. Inverse probability weighting was used to adjust for clinical and imaging baseline characteristics. Additionally, to explore the impact of MG (&amp;lt;40 mmHg vs. &amp;gt;40 mmHg) in patients with AVAi &amp;lt;0.6 cm2/m2 (PLG-SAS vs. HG-SAS) and to explore the impact of AVAi (&amp;lt;0.6 cm2/m2 vs. &amp;gt;0.6 cm2/m2) in patients with MG &amp;lt;40 mmHg (PLG-SAS vs MAS) we performed 2 different propensity score analyses. Patients were censored at the time of surgery. Results Overall, during 23 [IQR,10–47] months of follow-up 1003 patients died and 770 patients underwent aortic valve replacement. IPW-adjusted natural history was significantly better in patients with MAS, intermediate for patients with PLG-SAS and worst in patients with HG-SAS (59 vs. 47 vs. 41%, p&amp;lt;0.001, see Figure 1A). Furthermore, at equal MG (448 pairs), survival was significantly better in patients with MAS compared with PLG-SAS (54% vs. 39% p&amp;lt;0.001, see Figure 1B) and at equal AVAi (377 pairs), survival was significantly better in patients with PLG-SAS compared with HG-SAS (43% vs. 32% p&amp;lt;0.001, see Figure 1C). Conclusions In this large multicentric cohort, survival of PLG-SAS patients was better than that of HG-SAS patients and worse than that of MAS patients. Furthermore, with a comparable mean gradient, the smaller the calculated AVAi, the worse the prognosis whereas with a comparable AVAi, the higher the mean gradient, the worse the prognosis. Taking together, these data demonstrate that PLG-SAS is an intermediate form in the disease continuum, HG-SAS being the most malignant form of AS. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds National de la Recherche Scientifique (F.R.S.–FNRS)

Real-Time Integrated Learning and Decision Making for Cumulative Shock Degradation

Problem definition: Unexpected failures of equipment can have severe consequences and costs. Such unexpected failures can be prevented by performing preventive replacement based on real-time degradation data. We study a component that degrades according to a compound Poisson process and fails when the degradation exceeds the failure threshold. An online sensor measures the degradation in real time, but interventions are only possible during planned downtime. Academic/practical relevance: We characterize the optimal replacement policy that integrates real-time learning from the online sensor. We demonstrate the effectiveness in practice with a case study on interventional x-ray machines. The data set of this case study is available in the online companion. As such, it can serve as a benchmark data set for future studies on stochastically deteriorating systems. Methodology: The degradation parameters vary from one component to the next but cannot be observed directly; the component population is heterogeneous. These parameters must therefore be inferred by observing the real-time degradation signal. We model this situation as a partially observable Markov decision process (POMDP) so that decision making and learning are integrated. We collapse the information state space of this POMDP to three dimensions so that optimal policies can be analyzed and computed tractably. Results: The optimal policy is a state dependent control limit. The control limit increases with age but may decrease as a result of other information in the degradation signal. Numerical case study analyses reveal that integration of learning and decision making leads to cost reductions of 10.50% relative to approaches that do not learn from the real-time signal and 4.28% relative to approaches that separate learning and decision making. Managerial implications: Real-time sensor information can reduce the cost of maintenance and unplanned downtime by a considerable amount. The integration of learning and decision making is tractably possible for industrial systems with our state space collapse. Finally, the benefit of our model increases with the amount of data available for initial model calibration, whereas additional data are much less valuable for approaches that ignore population heterogeneity. Funding: This work was supported by the Fonds National de la Recherche Luxembourg [Grant AFR-12451704] and Philips. The work of C. Drent is supported by the Data Science Flagship framework, a cooperation between Eindhoven University of Technology and Philips. The research of M. Drent is supported by the National Research Fund of Luxembourg. J. Arts thanks the Netherlands Foundation for Scientific Research for funding this research. The work of S. Kapodistria is supported by the NWO Gravitation Project NETWORKS of the Dutch government. Supplemental Material: The online appendices are available at https://doi.org/10.1287/msom.2022.1149 .

Timing evolution of lobular breast cancer through phylogenetic analysis.

SummaryBackgroundLate distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood.MethodsWe retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients.FindingsThe data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients.InterpretationOur results provide insights into ILC evolution and offer potential paths for optimised ILC care.FundingThis work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

Perceived loneliness and the role of cultural and intergenerational belonging: the case of Portuguese first-generation immigrants in Luxembourg

The risk of loneliness for migrants, particularly in older age, has been documented across multiple studies. Migration is a life-changing transition. While often retaining links to their country of origin, an important developmental task for migrants is the establishment of bonds in the receiving country. Drawing on recent studies, I will explore the role of cultural and intergenerational belonging in order to identify both protective and risk factors regarding loneliness in middle and older age in a sample of first-generation immigrants from Portugal living in Luxembourg. The sample comprises N = 131 participants (51.9% female) between the ages of 41 and 80 (M = 56.08; SD = 7.80) who have on average spent M = 31.71 years (SD = 8.81) in Luxembourg and raised children in Luxembourg. They took part in the IRMA project (‘Intergenerational Relations in the Light of Migration and Ageing’) which was funded by the Fonds National de la Recherche Luxembourg. A standardised questionnaire assessed socio-demographic data, aspects of cultural belonging (i.e. cultural attachment to both countries, bicultural identity orientation, acculturative stress), intergenerational belonging (i.e. family cohesion, family conflict, perceived intergenerational value consensus) and perceived loneliness. Results showed that while cultural and intergenerational belonging were protective factors, the strongest predictors for participants’ perceived loneliness were cultural identity conflict and, even more so, intergenerational conflict. Our findings suggest that establishing roots and bonds in the host country is a protective factor against loneliness, whereas the feeling of not fitting in is a strong risk factor.

Ovarian tissue cryopreservation and transplantation in patients with central nervous system tumours.

Is there a possibility of reseeding cancer cells potentially present in frozen ovarian tissue from patients with central nervous system (CNS) tumours? Malignancy reseeding in cryopreserved ovarian tissue from 20 patients with CNS tumours was not detected by histology, immunohistochemistry (IHC), molecular biology or xenotransplantation. Ovarian metastasis potential has been documented in patients with leukaemia, borderline ovarian tumours, advanced breast cancer and Ewing sarcoma. However, data on the safety of transplanting frozen-thawed ovarian tissue from cancer patients with CNS tumours are still lacking. This prospective experimental study was conducted in an academic gynaecology research laboratory using cryopreserved ovarian cortex from 20 patients suffering from CNS tumours. Long-term (5 months) xenografting was performed in immunodeficient mice. Subjects enrolled in the study were suffering from one of six types of CNS tumours including medulloblastoma, ependymoma, primitive neuroectodermal tumours, astrocytoma, glioblastoma and germinoma. The presence of malignant cells was investigated with disease-specific markers for each patient in cryopreserved and xenografted ovarian tissue by histology, IHC via expression of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP), and reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) for quantification of GFAP and ENO2 gene amplification. Serial sections of cryopreserved and xenografted ovarian tissue from 20 patients showed no malignant cells by histology. All samples were negative for NSE and GFAP, although these neural markers were expressed extensively in the patients' primary tumours. Analysis by RT-ddPCR revealed no cancer cells detected in cryopreserved and xenografted ovarian fragments from subjects with astrocytoma, ependymoma, glioblastoma or medulloblastoma. Taken together, the study found no evidence of malignancy seeding in frozen-thawed and xenotransplanted ovarian tissue from patients affected by CNS cancers. This analysis cannot guarantee complete elimination of disseminated disease from all cryopreserved ovarian cortex, since we are unable to examine the fragments used for transplantation. This is the first study to be conducted in patients with CNS cancers undergoing ovarian tissue cryopreservation and transplantation, and clearly demonstrates no tumour seeding in their frozen-thawed and xenografted tissue. This information is vital for doctors to provide patients with meaningful and accurate advice on the possibilities and risks of ovarian tissue reimplantation. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique-the Excellence of Science (FNRS-EOS), number 30443682 awarded to M.-M.D. and T.Y.T.N., FNRS grant number 5/4/150/5 and FNRS-PDR Convention grant number T.0077.14 awarded to M.-M.D., grant 2018-042 from the Foundation Against Cancer awarded to A.C., and private donations (Ferrero, de Spoelberch). The authors declare no competing financial interests. N/A.

In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women.

Can human theca cells (TCs) be differentiated in vitro? It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, respectively. Results obtained from qPCR showed a significant increase (P < 0.05) in mRNA levels of Lhr in F2 (floating cells in G2) and G4, Cyp17a1 in G1 and F1 (floating cells in G1) and Hsd3b2 in G1, G2, G3 and G4. Immunohistochemistry confirmed expression of each enzyme involved in the steroidogenic pathway at the protein stage. However, apart from G1, all other groups exhibited a significant (P < 0.05) rise in the number of CD13-positive cells. There was also a significant increase (P < 0.05) in NOTCH1-positive cells in G3 and G4. Ultrastructure analyses by TEM showed a distinct difference between groups and also versus Day 0. A linear trend with time revealed a significant gain (q < 0.001) in DHEA concentrations in the medium during the culture period in G1, G2, G3 and G4. It also demonstrated a statistical increase (q < 0.001) in G2, G3 and G4 groups, but G1 remained the same throughout culture in terms of progesterone levels. N/A. Shorter periods of in vitro culture (e.g. 2, 4 and 6 days) could have led to increased concentrations of differentiated TCs in G2, G3 and G4. In addition, a group of cells cultured in BM and accompanied by COV434 cells would be necessary to understand their role in the differentiation process. Finally, while our results demonstrate that TCs can be differentiated in vitro from cells isolated from the cortical layer of postmenopausal ovaries, we do not know if these cells are differentiated from a subpopulation of precursor TCs present in ovarian cortex or ovarian SCs in general. It is therefore necessary to identify specific markers for precursor TCs in human ovaries to understand the origin of these cells. This is a promising step toward understanding TC ontogenesis in the human ovary. Moreover, in vitro-generated human TCs can be used for studies on drug screening, as well as to understand TC-associated pathologies, such as androgen-secreting tumors and polycystic ovary syndrome. This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C.A.A.; grant 5/4/150/5 awarded to M.M.D.; grant ASP-RE314 awarded to P.A.) and Foundation Against Cancer (grant 2018-042 awarded to A.C.). The authors declare no competing interests.

Editorial introductions

Editorial introductions

Lung immunoglobulin A immunity dysregulation in cystic fibrosis.

BackgroundIn cystic fibrosis (CF), recurrent infections suggest impaired mucosal immunity but whether production of secretory immunoglobulin A (S-IgA) is impaired remains elusive. S-IgA is generated following polymeric immunoglobulin receptor (pIgR)-mediated transepithelial transport of dimeric (d-)IgA and represents a major defence through neutralisation of inhaled pathogens like Pseudomonas aeruginosa (Pa).MethodsHuman lung tissue (n = 74), human sputum (n = 118), primary human bronchial epithelial cells (HBEC) (cultured in air-liquid interface) (n = 19) and mouse lung tissue and bronchoalveolar lavage were studied for pIgR expression, IgA secretion and regulation.FindingsIncreased epithelial pIgR immunostaining was observed in CF lung explants, associated with more IgA-producing plasma cells, sputum and serum IgA, especially Pa-specific IgA. In contrast, pIgR and IgA transport were downregulated in F508del mice, CFTR-inhibited HBEC, and CF HBEC. Moreover, the unfolded protein response (UPR) due to F508del mutation, inhibited IgA transport in Calu-3 cells. Conversely, pIgR expression and IgA secretion were strongly upregulated following Pa lung infection in control and F508del mice, through an inflammatory host response involving interleukin-17.InterpretationA complex regulation of IgA secretion occurs in the CF lung, UPR induced by CFTR mutation/dysfunction inhibiting d-IgA transcytosis, and Pa infection unexpectedly unleashing this secretory defence mechanism.FundingThis work was supported by the Forton's grant of the King Baudouin's Foundation, Belgium, the Fondazione Ricerca Fibrosi Cistica, Italy, and the Fonds National de la Recherche Scientifique, Belgium.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Spatiotemporal changes in mechanical matrisome components of the human ovary from prepuberty to menopause.

Phylogenetic reconstruction of breast cancer reveals two routes of metastatic dissemination associated with distinct clinical outcome.

BackgroundIn breast cancer (BC), axillary lymph node (ALN) involvement is one of the strongest adverse prognostic factors. However, it is unclear whether loco-regional lymph node deposits are effectively the root of secondary metastases or only an indicator of competence of the primary tumour to spread to distant organs.MethodsHere, we investigated the evolutionary trajectories of primary tumour, ALN and distant metastasis samples from 16 estrogen-receptor (ER)-positive lymph node-positive BC patients. Low-pass whole genome sequencing was performed to infer somatic copy number aberrations and the phylogenetic profiles for all patients were obtained.FindingsWe show that lymph nodes and distant metastases shared a common origin in only 25% of the cases highlighting that the predominant route of metastatic dissemination is the direct seeding of tumour cells from the primary tumour to distant organs, independently of lymph node metastasis. Noticeably, patients sharing a common origin significantly have worse prognosis.InterpretationOur results shed light on the routes on which tumour cells metastasize and their role in disease progression in ER-positive BC.FundingThis work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF), the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS) and from a grant of the Région Wallonne.

Editorial introductions

Editorial introductions

360-OR: A Novel Genetic Syndrome of Early-Onset Diabetes, Microcephaly, and Epilepsy Due to Homozygous YIPF5 Mutations

Neonatal diabetes is caused by single gene mutations affecting fundamental β-cell function pathways. 20% of cases of neonatal diabetes remain genetically unexplained. Our aim was to explore the genetic basis of a syndrome characterized by neonatal diabetes, microcephaly and epilepsy, present in 2 unrelated patients. The Turkish and Indian patients were born to consanguineous parents. Using whole genome sequencing, we found that they had homozygous likely deleterious variants (missense, p.(Ala181Val) and in-frame deletion p.(Lys106del)) in YIPF5. We then performed replication studies in 112 patients with neonatal diabetes by targeted next generation sequencing. These identified 2 homozygous YIPF5 mutations (p.(Trp218Arg) and p.(Ile98Ser)) in 3 patients (2 siblings) with early-onset diabetes, epilepsy and microcephaly. In functional studies, YIPF5 was expressed in human islets and fetal brain cortex, as evaluated by qPCR and in situ hybridization. Because YIPF5 is involved in endoplasmic reticulum (ER)-to-Golgi trafficking, we examined the impact of YIPF5 loss-of-function on β-cell survival during ER stress. YIPF5 was silenced in the human β-cell line EndoC-βH1 and in human islets. YIPF5 silencing sensitized β cells to apoptosis induced by the ER stressors thapsigargin and brefeldin A. The ER stressors enhanced CHOP, BiP and spliced XBP1 expression in YIPF5-depleted cells, indicating increased ER stress signaling. Expression of the proapoptotic proteins PUMA and DP5 was also enhanced by YIPF5 silencing. CHOP and DP5 knockdown partially protected YIPF5-deficient cells from apoptosis, suggesting that they mediate apoptosis. In conclusion, homozygous loss-of-function mutations in YIPF5 are a novel cause of early-onset diabetes, microcephaly and epilepsy. This syndrome unveils a critical role of YIPF5 and ER-to-Golgi trafficking in the function and survival of human β-cells and neurons. Disclosure M. Lytrivi: None. E. De Franco: Other Relationship; Self; Novo Nordisk Foundation. K.A. Patel: None. M. Igoillo Esteve: None. C. Cosentino: None. M.N. Wakeling: None. B. Haliloglu: None. M. Yildiz: None. T. Godbole: None. A.T. Hattersley: None. M. Cnop: None. Funding Fonds Erasme for Medical Research; European Union (667191); Fonds National de la Recherche Scientifique; Brussels-Capital Region Innoviris