Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos.

PURPOSE: Unpredictable genetic modifications and chromosomal aberrations following CRISPR/Cas9 administration hamper the efficacy of germline editing. Repair events triggered by double-strand DNA breaks (DSBs) besides non-homologous end joining and repair template-driven homology-directed repair have been insufficiently investigated in mouse. In this work, we are the first to investigate the precise repair mechanisms triggered by parental-specific DSB induction in mouse for paternal mutational correction in the context of an infertility-related mutation. METHODS: We aimed to correct a paternal 22-nucleotide deletion in Plcz1, associated with lack of fertilisation in vitro, by administrating CRISPR/Cas9 components during intracytoplasmic injection of Plcz1-null sperm in wild-type oocytes combined with assisted oocyte activation. Through targeted next-generation sequencing, 77 injected embryos and 26 blastomeres from seven injected embryos were investigated. In addition, low-pass whole genome sequencing was successfully performed on 17 injected embryo samples. RESULTS: Repair mechanisms induced by two different CRISPR/Cas9 guide RNA (gRNA) designs were investigated. In 13.73% (7/51; gRNA 1) and 19.05% (4/21; gRNA 2) of the targeted embryos, only the wild-type allele was observed, of which the majority (85.71%; 6/7) showed integrity of the targeted chromosome. Remarkably, for both designs, only in one of these embryos (1/7; gRNA 1 and 1/4; gRNA2) could repair template use be detected. This suggests that alternative repair events have occurred. Next, various genetic events within the same embryo were detected after single-cell analysis of four embryos. CONCLUSION: Our results suggest the occurrence of mosaicism and complex repair events after CRISPR/Cas9 DSB induction where chromosomal integrity is predominantly contained.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

[Feasibility study: The medical imaging as a tool for therapeutic education in radiotherapy]. / Étude de faisabilité : l'utilisation de l'imagerie médicale en éducation thérapeutique en radiothérapie.

PURPOSE: Assess the feasibility of a randomized controlled trial (RCT) exploring the use of medical imaging as a therapeutic education (TPE) intervention in external radiation therapy. MATERIALS AND METHODS: Experimental feasibility trial of "RCT" type carried out in a single-center, between November 2019 and March 2020, following adult patients treated by thoracic radiotherapy. In addition to the information usually given, the experimental group benefited from an intervention consisting in the visualization of their own medical images using the open-source software "Stone of Orthanc". RESULTS: Forty-nine patients were recruited with a refusal rate of 8.16% (4/49). 20 patients were withdrawn from the study for health reasons (COVID), 10 for medical reasons. All the remaining 15 participants completed the process. Although not significant, the experimental group showed a median gain in the perception of knowledge compared to the control group (+ 1.9 (1.6 - 2.2)) vs (+ 1.4 (1.4 - 1.8)), as well as a decrease in scores related to anxiety (- 3.0 (-4.5 - (-2.0)) vs - 1.0 (-5.0 - 0.0)) and emotional distress ((- 5.0 (- 7.5 - (- 3.5)) vs (- 2.0 (- 5.0 - (- 1.0)) A significant reduction (p=0.043) is observed for the depression score ((- 2.0 (-3.0 - (-1.5)) vs (0.0 (0.0 - 0.0)). CONCLUSION: This study demonstrates the feasibility of the project, with promising preliminary results. Some adaptations in order to conduct a larger-scale RCT are highlighted.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

The Abcc6a Knockout Zebrafish Model as a Novel Tool for Drug Screening for Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a multisystem ectopic mineralization disorder caused by pathogenic variants in the ABCC6 gene. Though complications of the disease can be treated, PXE itself remains currently intractable. A strategy for rapid and cost-effective discovery of therapeutic drugs would be to perform chemical compound screening using zebrafish, but this approach remains to be validated for PXE. In this paper, we validate a stable CRISPR/Cas9 abcc6a knockout zebrafish model-which has spinal column hypermineralization as its primary phenotypic feature-as a model system for compound screening in ectopic mineralization. We evaluated the anti-mineralization potential of five compounds, which had (anecdotal) positive effects reported in Abcc6 knockout mice and/or PXE patients. Abcc6a knockout zebrafish larvae were treated from 3 to 10 days post-fertilization with vitamin K1, sodium thiosulfate, etidronate, alendronate or magnesium citrate and compared to matching controls. Following alizarin red S staining, alterations in notochord sheath mineralization were semiquantified and found to largely congrue with the originally reported outcomes. Our results demonstrate that the use of this abcc6a knockout zebrafish model is a validated and promising strategy for drug discovery against ectopic mineralization.

Review of interactive digital solutions improving health literacy of personal cancer risks in the general public.

BACKGROUND: Health literacy is crucial in understanding the many risk factors for cancer. Low health literacy is associated with low adherence to medication, poor health status, and increased health care costs. Modern technology allows us to educate the general public on their risks. We focus herein on the available mobile applications and online web tools for the evaluation of cancer risk in the general public. METHODS: A systematic search was performed for cancer risk calculators mobile applications on both Google Play and the App Store and for online cancer risk calculators using Google, Bing, Yahoo! and Baidu. RESULTS: For mobile applications, out of 250 different apps found on GooglePlay, 16 Android applications were retained for evaluation in this review and for the AppStore, out of 10 different apps, 7 Android applications were retained for evaluation in this review. Only three apps were available for both Android and iOS systems. For web tools, a list of 20 tools was retained and evaluated. CONCLUSION: This review presents the most popular and prominent tools and their strengths and possible weaknesses are evaluated. We discuss not only its current state as it relates to general knowledge about cancer risks, but also barriers and future directions. It is imperative that as developers continue to create and improve such tools, health care providers remain aware of these efforts in order to properly guide patients towards appropriate resources and educate them on both their usefulness and limitations.

[Mental illnesses: the playground for new technologies]. / La médecine du futur. Maladies mentales : le terrain de jeux des nouvelles technologies.

The current health care crisis, induced by the coronavirus pandemic, is at the origin of significant global changes within our societies and profoundly modifies the health care sector as well, especially in the field of mental health. Nowadays, this latter is particularly poorly equipped in financial and human resources. Without major and immediate changes, the mental health sector will not be able to cope with the expected exponential rise of care needs, exacerbated by the rapid deterioration of mental health in the general population and among health care providers. We intend to illustrate the potential role and benefit of new technologies, able to solve the imbalance. Without any possible doubt, the health care crisis has provided a formidable momentum for their arousal, but we still have to determine their accessibility, feasibility, efficacy and efficiency by running controlled clinical trials.

La crise sanitaire liée au coronavirus est à l'origine de chamboulements majeurs au niveau sociétal, qui influencent, par la même occasion, l'organisation des soins de santé et les besoins, en particulier en santé mentale. Aujourd'hui, le domaine de la santé mentale est particulièrement mal équipé en ressources financières et humaines. Sans changements majeurs immédiats, la société ne pourra pas faire face à une croissance vertigineuse attendue des demandes, croissance liée à la détérioration accélérée de la santé mentale dans la population générale mais aussi parmi les soignants. Nous faisons le point sur les nouvelles technologies qui peuvent résoudre, au moins en partie, ce déséquilibre, tout en signalant que, même si la conjoncture sanitaire actuelle a indéniablement accéléré leur irruption dans le domaine, il faudra en objectiver l'accessibilité, la faisabilité, l'efficacité et l'efficience par la conduite d'essais cliniques contrôlés.

[Radiomics and automation in radiotherapy]. / Radiomique et automatisation en radiothérapie : un duo gagnant !

Cancer incidence is steadily progressing worldwide, in parallel with the aging of the population. Workload is increasing constantly, especially in the fields of oncology and radiotherapy. This is particularly worrysome, as there is a general shortage of skilled professionals in the field (for example in medical physics). Moreover, every single patient does represent an enormous amount of data issued from a wide range of sources. This is especially true as far a medical imaging is concerned. Extraction of morphological data (anatomical location and extent of the tumour) and functional data (tumour biology and metabolism in general) becomes laborious. Moreover, images contain information which cannot be discerned by the human eye. Therefore, to handle shortage of human resources and transform this enormous amount of data automatically, artificial intelligence becomes a «must have¼. We intend to highlight the growing importance of radiomics as a cornerstone in automation of processes in radiotherapy, especially for treatment planification and a more personalized individualized treatment approach.

L'incidence du cancer augmente chaque année suivant de très près l'augmentation de la moyenne d'âge de la population. La charge de travail ne fait qu'augmenter en oncologie, y compris en radiothérapie. Il devient difficile de recruter certains professionnels dans le secteur (comme par exemple, des physiciens). À ce manque, vient s'ajouter l'afflux massif de données pour chaque patient, provenant d'une multitude de sources possibles et, en particulier, la quantité et la complexité des informations contenues dans les différents examens d'imagerie. L'extraction des données structurelles (anatomie et extension de la maladie) et fonctionnelles (biologie et activité métabolique tumorale au sens large) devient laborieuse. De plus,certains éléments contenus dans l'image numérique ne sont tout simplement plus accessibles à nos capacités de perception visuelle. La pénurie annoncée en professionnels experts ainsi que la complexité grandissante de l'analyse de l'image méritent l'apport de l'intelligence artificielle. Nous allons faire le point de l'impact attendu de la «radiomique¼ qui permet l'automatisation des processus, en particulier pour la préparation et l'individualisation des traitements en radiothérapie.

[Palliation by radiation is also our business !] / La radiothérapie palliative, c'est aussi notre rayon !

Radiotherapy (RT), both with a curative and a palliative intent, is one of the cornerstones of oncological treatments. A variety of symptoms linked to cancer can be relieved with RT (such as pain, bleeding, compression exerted by a tumour lesion…). Very often, palliative RT is proposed when other medical treatments (painkillers, morphine…) are no longer efficient, or the patient does not tolerate them anymore. Palliative RT is an integral part of the global supportive oncological care. Indeed, patients' wishes and prognosis are taken into account in each and every step of the treatment pathway. Every treatment deserves an individualized approach and benefits from the best available techniques.

La radiothérapie, à la fois à visée curative et palliative, est l'un des piliers des traitements oncologiques. Une multitude de symptômes liés au cancer (douleurs, saignements, diverses conséquences liées à une compression exercée par une lésion tumorale…) peuvent être soulagés grâce à une radiothérapie palliative (RTP). Bien souvent, la RTP est proposée lorsque les traitements médicamenteux dits «classiques¼ ne font plus suffisamment effet ou si le patient ne les tolère plus (antidouleurs, morphine…). La RTP fait partie intégrante des soins oncologiques de support. En effet, le pronostic du patient, ainsi que ses souhaits, sont pris en compte à chacune des étapes qui constituent le trajet de soins, y compris en RTP. Ainsi, chaque traitement est individualisé et bénéficie des meilleures techniques disponibles.

[Precision medicine in oncology : where do we stand ?] / Médecine de précision en oncologie : où en sommes-nous ?

The «one size fits all¼ approach is seriously challenged by rapid progression of medical knowledge, especially in the field of individual genome expression. It is currently known that the anti-tumour effect of a given treatment and possible side effects at the level of healthy tissues, can at least partly be predicted and explained by individual variations of gene expression. However, most of us realize that these differences in response are also linked to a variety of other individual characteristics, such as for example the environment and socio-economic factors. Without any possible doubt, there are multiple problems (technical, administrative, financial, cultural and ethical) to be solved, before we witness the real irruption of precision medicine and its holistic individualized approach in our daily oncological practice. It has to start with an international effort, disregarding borders of individual countries, in order to obtain very large amounts of data (with a high degree of variability to avoid bias). This holistic approach, at both societal and individual levels, is the entrance door for a personalized approach in care, whether this is curative, predictive or preventive.

Le concept du traitement «taille unique¼ est sérieusement remis en question par la progression accélérée des connaissances du rôle prédictif du génome individuel, tant en matière de la réponse tumorale à un traitement, qu'en ce qui concerne l'apparition d'effets secondaires au niveau des tissus sains. Cette réponse au traitement ne dépend pas simplement de l'expression de quelques gènes, ni d'ailleurs du génome individuel entier. Elle est également influencée par une multitude d'autres facteurs, ce qui requiert une approche holistique prenant en compte, par exemple, l'environnement et les facteurs socio-économiques. Il y a indubitablement des problèmes techniques, administratifs, financiers, culturels et éthiques (protection de la vie privée) à surmonter avant que cette médecine de précision ne soit largement disponible en pratique oncologique journalière. Il faut surtout une collaboration allant largement au-delà des frontières géographiques d'un pays, afin d'assurer une manne suffisante et très variable de données, pour éliminer autant que possible les différents biais. Cette approche holistique, tant au niveau sociétal qu'individuel, ouvre la porte à la personnalisation des soins oncologiques dans le domaine curatif, prédictif et préventif.

[How to improve radiotherapy ? Technological developments and radiotherapy of tomorrow]. / Comment améliorer la radiothérapie ? Évolutions technologiques et radiothérapie de demain.

Radiotherapy established itself in the 20th century as an essential modality in the fight against cancer. The major technological advances of the last decades have allowed a considerable improvement in the therapeutic window. They have also paved the way for stereotactic radiotherapy and new indications. The aim of this article is to enable readers to understand external radiotherapy in 2021 and to understand the challenges of tomorrow. Three areas of improvement in the discipline will be described, the optimization of the prescribed therapeutic dose, the improvement of the distribution of this dose and, finally, the better understanding of radiobiology. For each of these axes, the current implications will be described as well as those which could/should have a major impact on the radiotherapy. FLASH radiotherapy will also be discussed.

La radiothérapie s'est installée au 20ème siècle comme une modalité incontournable dans la lutte contre le cancer. Les avancées technologiques majeures des dernières décennies ont permis une amélioration considérable de la fenêtre thérapeutique. Elles ont également ouvert la voie à la radiothérapie stéréotaxique et à de nouvelles indications. Cet article a pour objectif de permettre aux lecteurs de comprendre la radiothérapie externe en 2021 et d'en appréhender les enjeux de demain. Trois axes d'amélioration de la discipline seront décrits, l'optimisation de la dose thérapeutique prescrite, l'amélioration de la distribution de cette dose et, finalement, la meilleure compréhension de la radiobiologie. Pour chacun de ces axes, des implications actuelles seront données ainsi que celles qui pourraient/ devraient avoir un impact sur la radiothérapie future. La radiothérapie FLASH sera, par exemple, abordée.

[Dental management of head and neck irradiated patients]. / Prise en charge dentaire des patients irradiés en région cervico-faciale.

The oncological management of head and neck tumours is well known and standardized. Radiotherapy is one of the effective tools. However, it induces major changes in healthy tissues: teeth, gums, mucous membranes, salivary glands and bones. Some, like mucositis, are immediate and often reversible; others, like hyposialia or fibrosis, are late effects and often irremediable. These changes greatly affect oral health and make its management more complex. Dental management also becomes a capital element of the care path but, unfortunately, often remains neglected by the patient but also by some practitioners. It concerns all the stages of the clinical course: initial assessment, cancer treatment itself and long-term follow-up. If neglected, the patient's quality of life will be affected and complications, sometimes serious, such as osteoradionecrosis, may occur. Specific care recommendations for maintaining oral health are mentioned, especially for those patients requiring oral cavity irradiation.

La prise en charge carcinologique des tumeurs cervico-faciales est bien connue et codifiée. La radiothérapie fait partie des outils efficaces proposés. Elle entraîne cependant de profondes modifications tissulaires : dents, gencives, muqueuses, glandes salivaires, os. Certaines, comme la mucite, sont immédiates, et souvent réversibles; d'autres, comme l'hyposialie ou la fibrose, s'installent tardivement et souvent définitivement. Ces remaniements altèrent fortement la santé bucco-dentaire et rendent la prise en charge plus complexe. L'approche dentaire devient ainsi un élément capital du trajet de soins. Elle reste, malheureusement, souvent délaissée par le patient lui-même, mais aussi parfois par le praticien. Cette prise en charge concerne toutes les étapes du parcours : bilan initial, traitement carcinologique en soi et suivi à long terme. Si négligée, la qualité de vie du patient sera affectée et des complications, parfois graves, telle l'ostéoradionécrose, peuvent survenir. Sont évoquées ici des recommandations spécifiques de prise en charge bucco-dentaire dans le décours d'une irradiation portant sur la cavité buccale.

[Pharma industry reinvents itself in the turmoil. Part 2. Opportunities]. / Dans la tourmente, l'industrie pharma se réinvente. Partie 2. Les opportunités.

In the first article we described how pharmaceutical industry is facing major challenges in general. In this second part, we intend to focus on new concepts and developments, which might sound «futuristic¼ for most of us. However, this seems definitely «work in rapid progress¼, especially concerning, for example, the use of the pharmacogenome (prerequisite for treatment personalization), «clever¼ medication, 3D printing and labelling with QR code. The future is bright but we cannot omit to quote the ever growing role of incumbent actors, both in the fields of medication production as well as distribution.

Dans la première partie, nous avons évoqué les changements généraux auxquels l'industrie pharmaceutique doit faire face. Dans cette seconde partie, nous passons en revue quelques concepts et développements qui, pour beaucoup d'entre nous, semblent «futuristes¼, mais dont les grandes lignes se dessinent déjà. On évoquera brièvement quelques exemples comme l'analyse du pharmacogénome (antichambre pour la prescription personnalisée), les médicaments «intelligents¼, l'impression 3D des médicaments et leurs marquages. On ne peut pas parler de l'avenir de l'industrie pharmaceutique, sans évoquer le rôle grandissant de nouveaux acteurs dans les chaines de production et de distribution.

[Pharma industry reinvents itself in the turmoil. Part 1. Challenges]. / La médecine du futur. Dans la tourmente, l'industrie pharma se réinvente. Partie 1 : Les défis.

The pharmaceutical industry faces, as many other public and private sectors, a significant deficit in trust (medication considered too expensive, not always readily available, even if it is deemed essential, lack of financial transparency). Moreover, we are more than very surprised by the lack of efficiency (colossal investments, compared to the modest final output). Not a single human activity sector can afford the luxury of such a situation. In these moments of doubts, industrial leaders are pushed to reinvent the whole sector, starting from research and development, through production and commercialization, to finish with retail. They are massively investing in new disruptive technologies (artificial intelligence, connected health, big data), redesigning the way clinical trials are elaborated and performed.

Le secteur industriel pharmaceutique, comme beaucoup d'autres, fait face à un déficit en confiance de la part des consommateurs (médicaments trop chers, ruptures de stock de certains médicaments considérés comme essentiels, manque de transparence financière). De plus, il pèche singulièrement en efficience (investissements colossaux par rapport aux résultats finalement obtenus). Aucun secteur industriel ne peut se permettre un tel état de fait. Ce moment de doute, exacerbé par la crise sanitaire, pousse les capitaines industriels à repenser totalement le secteur. Pour cela, ils embarquent résolument les nouvelles technologies dans leurs portfolios, en les incorporant dans toute la chaîne d'activité, en partant du domaine de la recherche et du développement, en passant par la fabrication, et en terminant par la commercialisation et la distribution. Ils rénovent au passage également la manière dont les essais cliniques sont élaborés et conduits en faisant appel au «big data¼, à la médecine connectée et à l'intelligence artificielle.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences.

STUDY QUESTION: What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? SUMMARY ANSWER: POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. WHAT IS KNOWN ALREADY: Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. STUDY DESIGN, SIZE, DURATION: The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS: A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. LIMITATIONS, REASONS FOR CAUTION: One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

[Thinking differently about health care after COVID-19]. / Repenser les soins de santé post-COVID-19.

The health crisis linked to the coronavirus pandemic (COVID-19) has forced society and hospitals in particular to adapt and reform. Teamwork between hospitals, even beyond the networks, helped them to deal with the crisis. The medical and nursing staff had to learn to work differently and differentiate urgent from non-urgent care. But the patient also had to change his/her behaviour. Access to hospitals has been divided between a separate COVID and non-COVID route in order to avoid contamination. Telemedicine has become a daily way of communicating between doctors and patients. Telephone consultations have been set up with reimbursement by social security. However, these actions and innovations should not end with the crisis but, on the contrary, be a lever to rethink the role of hospitals, and our health care system more generally.

La crise sanitaire liée à la pandémie du coronavirus (COVID-19) a obligé la société, et les hôpitaux en particulier, à s'adapter et se réformer. Le travail en équipe entre hôpitaux, même au-delà des réseaux, a permis de faire face à la crise. Le corps médical et infirmier a dû apprendre à travailler différemment et faire la distinction entre les soins urgents et non urgents. Mais le patient aussi a dû changer ses comportements. L'accès aux hôpitaux s'est vu diviser entre un trajet COVID et non-COVID, bien distincts, afin d'éviter des contaminations. La télémédecine est devenue un moyen quotidien de communiquer entre le monde médical et les patients. Des consultations téléphoniques ont été instaurées avec, à la clef, un remboursement par l'INAMI. Cependant, ces actions et innovations ne devraient pas se terminer avec la crise liée à la COVID-19, mais, au contraire, être un levier pour repenser le rôle des hôpitaux, et notre système de soins de santé plus globalement.

[The COVID-19 pandemic : a real opportunity for digital health]. / La pandémie COVID-19 : une formidable opportunité pour les technologies digitales.

Nowadays, we are facing a global health crisis. The infectious agent, the virus SARS-CoV-2, has some clinical and pathological characteristics which have been described extensively throughout published medical literature. The pandemic outbreak arises in a very particular period. Never before, our political disorganization and lack of collaboration has been highlighted as it was during the present health care crisis. Our health care system is shaking because of the lack of sufficient human and financial resources. However, technological changes, and especially remote health (teleconsultations and remote monitoring) are disrupting the whole ecosystem. We intend to illustrate that the COVID outbreak offers a unique opportunity to accelerate acceptance of these rapid technological changes, which are anyway unavoidable. Teleconsultations and remote monitoring, which both appeared as a devil out of the box from nowhere, at least for some care providers in the health care landscape, are there to stay.

Nous faisons face à une crise sanitaire mondiale. L'agent pathogène incriminé, le SARS-CoV-2, se distingue des autres pathologies respiratoires par différents aspects cliniques et pathologiques, qui ont largement fait l'objet de multiples publications. Cette pandémie apparaît à une époque cruciale pour notre système des soins de santé. Elle a mis dans la lumière nos errements politiques et notre quasi-incapacité à gérer cette crise mondiale «ensemble¼. Elle a exacerbé les problèmes de financement des soins et des ressources humaines, y compris dans les pays nantis. La mise en place, dans l'urgence, de la technologie connectée, en particulier les consultations virtuelles, amène par ailleurs des changements disruptifs majeurs et indispensables dans l'écosystème des soins. Nous voulons démontrer que cette irruption brutale de la technologie connectée pourrait bien aider le monde des soins à aborder, et enfin accepter, ces changements devenus incontournables. La télémédecine, surgie de nulle part (quasiment du jour au lendemain pour certains prestataires) pourrait bien être l'exemple d'une percée sans retour en arrière vers un nouvel écosystème.

Germline nuclear transfer in mice may rescue poor embryo development associated with advanced maternal age and early embryo arrest.

STUDY QUESTION: Can pronuclear transfer (PNT) or maternal spindle transfer (ST) be applied to overcome poor embryo development associated with advanced maternal age or early embryo arrest in a mouse model? SUMMARY ANSWER: Both PNT and ST may have the potential to restore embryonic developmental potential in a mouse model of reproductive ageing and embryonic developmental arrest. WHAT IS KNOWN ALREADY: Germline nuclear transfer (NT) techniques, such as PNT and ST, are currently being applied in humans to prevent the transmission of mitochondrial diseases. Yet, there is also growing interest in the translational use of NT for treating infertility and improving IVF outcomes. Nevertheless, direct scientific evidence to support such applications is currently lacking. Moreover, it remains unclear which infertility indications may benefit from these novel assisted reproductive technologies. STUDY DESIGN, SIZE, DURATION: We applied two mouse models to investigate the potential of germline NT for overcoming infertility. Firstly, we used a model of female reproductive ageing (B6D2F1 mice, n = 155), with ages ranging from 6 to 8 weeks (young), 56 (aged) to 70 weeks (very-aged), corresponding to a maternal age of <30, ∼36 and ∼45 years in humans, respectively. Secondly, we used NZB/OlaHsd female mice (7-14 weeks, n = 107), as a model of early embryo arrest. This mouse strain exhibits a high degree of two-cell block. Metaphase II (MII) oocytes and zygotes were retrieved following superovulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian reserve was assessed by histological analysis in the reproductive-aged mice. Mitochondrial membrane potential (△Ψm) was measured by JC-1 staining in MII oocytes, while spindle-chromosomal morphology was examined by confocal microscopy. Reciprocal ST and PNT were performed by transferring the meiotic spindle or pronuclei (PN) from unfertilised or fertilised oocytes (after ICSI) to enucleated oocytes or zygotes between aged or very-aged and young mice. Similarly, NT was also conducted between NZB/OlaHsd (embryo arrest) and B6D2F1 (non-arrest control) mice. Finally, the effect of cytoplasmic transfer (CT) was examined by injecting a small volume (∼5%) of cytoplasm from the oocytes/zygotes of young (B6D2F1) mice to the oocytes/zygotes of aged or very-aged mice or embryo-arrest mice. Overall, embryonic developmental rates of the reconstituted PNT (n = 572), ST (n = 633) and CT (n = 336) embryos were assessed to evaluate the efficiency of these techniques. Finally, chromosomal profiles of individual NT-generated blastocysts were evaluated using next generation sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Compared to young mice, the ovarian reserve in aged and very-aged mice was severely diminished, reflected by a lower number of ovarian follicles and a reduced number of ovulated oocytes (P < 0.001). Furthermore, we reveal that the average △Ψm in both aged and very-aged mouse oocytes was significantly reduced compared to young mouse oocytes (P < 0.001). In contrast, the average △Ψm in ST-reconstructed oocytes (very-aged spindle and young cytoplast) was improved in comparison to very-aged mouse oocytes (P < 0.001). In addition, MII oocytes from aged and very-aged mice exhibited a higher rate of abnormalities in spindle assembly (P < 0.05), and significantly lower fertilisation (60.7% and 45.3%) and blastocyst formation rates (51.4% and 38.5%) following ICSI compared to young mouse oocytes (89.7% and 87.3%) (P < 0.001). Remarkably, PNT from zygotes obtained from aged or very-aged mice to young counterparts significantly improved blastocyst formation rates (74.6% and 69.2%, respectively) (P < 0.05). Similarly, both fertilisation and blastocyst rates were significantly increased after ST between aged and young mice followed by ICSI (P < 0.05). However, we observed no improvement in embryo development rates when performing ST from very-aged to young mouse oocytes following ICSI (P > 0.05). In the second series of experiments, we primarily confirmed that the majority (61.8%) of in vivo zygotes obtained from NZB/OlaHsd mice displayed two-cell block during in vitro culture, coinciding with a significantly reduced blastocyst formation rate compared to the B6D2F1 mice (13.5% vs. 90.7%; P < 0.001). Notably, following the transfer of PN from the embryo-arrest (NZB/OlaHsd) zygotes to enucleated non-arrest (B6D2F1) counterparts, most reconstructed zygotes developed beyond the two-cell stage, leading to a significantly increased blastocyst formation rate (89.7%) (P < 0.001). Similar findings were obtained after implementing ST between NZB/OlaHsd and B6D2F1 mice, followed by ICSI. Conversely, the use of CT did not improve embryo development in reproductive-age mice nor in the embryo-arrest mouse model (P > 0.05). Surprisingly, chromosomal analysis revealed that euploidy rates in PNT and ST blastocysts generated following the transfer of very-aged PN to young cytoplasts and very-aged spindles to young cytoplasts were comparable to ICSI controls (with young mouse oocytes). A high euploidy rate was also observed in the blastocysts obtained from either PNT or ST between young mice. Conversely, the transfer of young PN and young spindles into very-aged cytoplasts led to a higher rate of chromosomal abnormalities in both PNT and ST blastocysts. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The limited number of blastocysts analysed warrants careful interpretation. Furthermore, our observations should be cautiously extrapolated to humans given the inherent differences between mice and women in regards to various biological processes, including centrosome inheritance. The findings suggest that ST or PNT procedures may be able to avoid aneuploidies generated during embryo development, but they are not likely to correct aneuploidies already present in some aged MII oocytes. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study to evaluate the potential of PNT and ST in the context of advanced maternal age and embryonic developmental arrest in a mouse model. Our data suggest that PNT, and to a lesser extent ST, may represent a novel reproductive strategy to restore embryo development for these indications. STUDY FUNDING/COMPETING INTEREST(S): M.T. is supported by grants from the China Scholarship Council (CSC) (Grant no. 201506160059) and the Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF) (Grant no. 01SC2916 and no. 01SC9518). This research is also supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051017N, G051516N and G1507816N). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

[Medical imaging professionals and related specialties : a questioning is essential!] / La médecine du futur. Professionnels de l'imagerie médicale et spécialités annexées : une remise en question s'impose.

Nowadays, we are facing an overwhelming amount of public announcements concerning the rise of artificial intelligence (AI) in the world of medical imaging (including radiology, nuclear medicine and radiotherapy). While most of the applications are still limited to specific niches, there is a general trend to build real transversal platforms. Multiple industrial players, in collaboration with the clinicians in the field, are striving to build those platforms in order to offer plenty of use cases of AI for several purposes and needs (screening/detection, diagnosis and prediction). It is already undeniable that AI far exceeds human capabilities in terms of resolution, speed of image analysis and efficiency. Negative attitudes and skepticism from concerned professionals should be banned. Colla¬boration with data scientists and engineers for the large scale development and implementation should be pushed forward for the benefit of both patients and payers.

S'il y a bien un domaine où les annonces pleuvent en matière de développement de l'intelligence artificielle (IA), c'est le secteur de l'imagerie médicale au sens large du terme (regroupant la radiologie, la médecine nucléaire et la radiothérapie). Les applications, encore souvent uti¬lisées dans des niches précises, ont tendance à devenir beaucoup plus transversales. De multiples acteurs indus¬triels, en partenariat avec les utilisateurs, s'évertuent à construire de réelles plateformes qui offrent aux cliniciens une multitude d'applications utilisables pour combler plusieurs types de demandes et besoins (détection, diagnostic et prédiction). Il est indéniable que la capacité de l'IA dépasse largement nos capacités humaines en matière de résolution de l'image, de rapidité et d'efficience de lecture et d'analyse. Une attitude de négation ou de scepticisme de la part des professionnels du secteur n'est plus de mise. Ils doivent, sans attendre, collaborer avec les spécialistes data et les ingénieurs au développement à large échelle de l'IA en imagerie médicale et ce, au profit des patients et des payeurs.