The first section of this thesis aims to provide an international perspective on the characterisation of patients with axial spondyloarthritis. Through different aspects of the classification... Show moreThe first section of this thesis aims to provide an international perspective on the characterisation of patients with axial spondyloarthritis. Through different aspects of the classification criteria, we review the importance of global applicability of these criteria. As classification criteria ensure the same patients are selected for participation in clinical trials worldwide, global applicability would allow for direct comparisons between studies executed in different geographical regions. Likewise, standardised assessment and reporting of results from clinical trials allows for direct comparisons between studies investigating different treatments, or identical treatments in populations from a different ethnicity or background, which is debated in the second part of this thesis. Herein, we describe the process of the development of the core set for axial spondyloarthritis, specifically by updating the domains of the ASAS-OMERACT core set for ankylosing spondylitis. In the final part of this thesis, we discuss health-related quality of life and work and activity impairments in patients with chronic back pain suspected of axial spondyloarthritis. Show less
Stamatiadis, P.; Cosemans, G.; Boel, A.; Menten, B.; Sutter, P. de; Stoop, D.; ... ; Heindryckx, B. 2022
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... Show moreSTUDY 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 similar to 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. Show less
Navarro-Compan, V.; Boel, A.; Boonen, A.; Mease, P.J.; Dougados, M.; Kiltz, U.; ... ; Heijde, D. van der 2022
Objectives: To define the instruments for the Assessment of SpondyloArthritis international Society-Outcomes Measures in Rheumatology (ASAS-OMERACT) core domain set for axial spondyloarthritis ... Show moreObjectives: To define the instruments for the Assessment of SpondyloArthritis international Society-Outcomes Measures in Rheumatology (ASAS-OMERACT) core domain set for axial spondyloarthritis (axSpA). Methods: An international working group representing key stakeholders selected the core outcome instruments following a predefined process: (1) identifying candidate instruments using a systematic literature review; (2) reducing the list of candidate instruments by the working group, (3) assessing the instruments' psychometric properties following OMERACT filter 2.2, (4) selection of the core instruments by the working group and (5) voting and endorsement by ASAS. Results: The updated core set for axSpA includes seven instruments for the domains that are mandatory for all trials: Ankylosing Spondylitis Disease Activity Score and Numerical Rate Scale (NRS) patient global assessment of disease activity, NRS total back pain, average NRS of duration and severity of morning stiffness, NRS fatigue, Bath Ankylosing Spondylitis Function Index and ASAS Health Index. There are 9 additional instruments considered mandatory for disease-modifying antirheumatic drugs (DMARDs) trials: MRI activity Spondyloarthritis Research Consortium of Canada (SPARCC) sacroiliac joints and SPARCC spine, uveitis, inflammatory bowel disease and psoriasis assessed as recommended by ASAS, 44 swollen joint count, Maastricht Ankylosing Spondylitis Enthesitis Score, dactylitis count and modified Stoke Ankylosing Spondylitis Spinal Score. The imaging outcomes are considered mandatory to be included in at least one trial for a drug tested for properties of DMARD. Furthermore, 11 additional instruments were also endorsed by ASAS, which can be used in axSpA trials on top of the core instruments. Conclusions: The selection of the instruments for the ASAS-OMERACT core domain set completes the update of the core outcome set for axSpA, which should be used in all trials. Show less
Boel, A.; Lunteren, M. van; Lopez-Medina, C.; Sieper, J.; Heijde, D. van der; Gaalen, F.A. van 2022
Background: A positive family history (PFH) of spondyloarthritis (SpA) consists of five SpA-related entities, of which a PFH of axial spondyloarthritis (axSpA) is most common in European patients... Show moreBackground: A positive family history (PFH) of spondyloarthritis (SpA) consists of five SpA-related entities, of which a PFH of axial spondyloarthritis (axSpA) is most common in European patients with axSpA. Moreover, a PFH of axSpA is associated with human leucocyte antigen B27 (HLA-B27) positivity in these patients. It is unknown if this holds true in patients with axSpA in other parts of the world. Objective: To investigate the geographical prevalence of a PFH of SpA and its association with HLA-B27 positivity in patients with axSpA worldwide. Methods: Cross-sectional analyses included patients from the ASAS peripheral involvement in Spondyloarthritis (PerSpA) study from 24 countries worldwide with an axSpA diagnosis, known HLA-B27 status and family history. Logistic regression models were built to assess the effect of HLA-B27 status on the occurrence of PFH. This was repeated for each of the five SpA entities in a PFH. Results: Among 2048 patients, axSpA was the most common SpA entity in a PFH in all geographical regions (Asia 28%, Europe and North America 27%, Latin America 20%, Middle East and North Africa 41%). A PFH of axSpA was associated with HLA-B27 positivity in Asia (OR 4.19), Europe and North America (OR 2.09) and Latin America (OR 3.95), but not in the Middle East and North Africa (OR 0.98), which has a lower prevalence of HLA-B27 positivity. A PFH of other SpA entities was less prevalent and not consistently associated with HLA-B27 positivity. Conclusion: In patients with axSpA worldwide, axSpA was consistently the most common SpA entity in a family history and was associated with HLA-B27 positivity in all geographical regions but one. Show less
Boel, A.; Navarro-Compan, V.; Boonen, A.; Mease, P.; Kiltz, U.; Dougados, M.; ... ; Heijde, D. van der 2021
Objective. Advances in the field of axial spondyloarthritis (axSpA) and the methodology to develop core sets have led the Assessment of SpondyloArthritis international Society (ASAS) group to... Show moreObjective. Advances in the field of axial spondyloarthritis (axSpA) and the methodology to develop core sets have led the Assessment of SpondyloArthritis international Society (ASAS) group to update the ASAS-Outcomes in Rheumatology (OMERACT) core set. An important aspect was to ensure it would be applicable to the entire spectrum of axSpA. The first step was to define the most relevant disease domains.Methods. A 3-round Delphi survey was conducted to gather opinions of 188 patients and 188 axSpA experts to define the most relevant disease domains to be included in the core set. The Delphi survey evaluated 2 separate research settings: (1) studies assessing symptom-modifying therapies; and (2) studies evaluating disease-modifying therapies. Importance of the domains was rated on a 1-9 Likert scale. A domain was considered for inclusion if, for both stakeholder groups, >= 70% of participants scored the domain as critical (7-9) and <= 15% scored it as not important (1-3) after 3 rounds.Results. A total of 132 (70%) patients and 135 (72%) experts completed at least 1 round. After 3 rounds, 7 domains (pain, physical function, stiffness, disease activity, mobility, overall functioning and health, peripheral manifestations) were selected for the symptom-modifying therapies setting. For the disease-modifying therapies setting, 6 domains (physical function, disease activity, mobility, structural damage, extramusculoskeletal manifestations, peripheral manifestations) were selected. All domains selected by experts were also selected by patients. Patients selected all offered domains except emotional function.Conclusion. This study provides the domains selected by patients and axSpA experts that should be considered for the core set for axSpA. Show less
Boel, A.; Navarro-Compan, V.; Heijde, D. van der 2021
Objectives Aim of this study was to assess test-retest reliability of candidate instruments for the mandatory domains of the Assessment of Spondyloarthritis international Society (ASAS)-Outcome... Show moreObjectives Aim of this study was to assess test-retest reliability of candidate instruments for the mandatory domains of the Assessment of Spondyloarthritis international Society (ASAS)-Outcome Measures in Rheumatology core set for axial spondyloarthritis (axSpA). Methods Screening and baseline data from COAST-V, COAST-X and RAPID-axSpA was used to evaluate test-retest reliability of each candidate instrument for the mandatory domains (disease activity, pain, morning stiffness, fatigue, physical function, overall functioning and health). A maximum time interval of 28 days between both visits was used for inclusion in this study. Test-retest reliability was assessed by intraclass correlation coefficient (ICC). Bland and Altman plots provided mean difference and 95% limits of agreement, which were used to calculate the smallest detectable change (SDC). Data were analysed for radiographic and non-radiographic axSpA separately. Results Good reliability was found for Ankylosing Spondylitis Disease Activity Score (ICC 0.79, SDC 0.6), C reactive protein (ICC 0.72-0.79, SDC 12.3-17.0), Bath Ankylosing Spondylitis Functional Index (ICC 0.87, SDC 1.1) and 36-item Short-Form Health Survey (ICC Physical Component Summary 0.81, SDC 4.7, Mental Component Summary 0.80, SDC 7.3). Moderate reliability was found for Bath Ankylosing Spondylitis Disease Activity Index (ICC 0.72, SDC 1.1), patient global assessment (ICC 0.58, SDC 1.5), total back pain (ICC 0.64, SDC 1.3), back pain at night (ICC 0.67, SDC 1.3), morning stiffness (ICC 0.52-0.63, SDC 1.5-2.2), fatigue (ICC 0.65, SDC 1.3) and ASAS-Health Index (ICC 0.74, SDC 2.5). Reliability and SDC for the radiographic and non-radiographic axSpA subgroups were similar. Conclusion Overall reliability was good, and comparable levels of reliability were found for patients with radiographic and non-radiographic axSpA, even though most instruments were developed for radiographic axSpA. Composite measures showed higher reliability than single-item measures in assessing disease activity in patients with axSpA. Show less
Boel, A.; Lopez-Medina, C.; Heijde, D.M.F.M. van der; Gaalen, F.A. van 2021
Objective: Age at onset is useful in identifying chronic back patients at an increased risk of axial SpA (axSpA). However, the majority of data on which the criterion of age at onset. Methods:... Show moreObjective: Age at onset is useful in identifying chronic back patients at an increased risk of axial SpA (axSpA). However, the majority of data on which the criterion of age at onset. Methods: Analyses were applied to patients from 24 countries across the world with an axSpA diagnosis and known age at onset of axial complaints. Cumulative probability plots were used to display the cumulative distribution of age at onset of axial symptoms. Linear regression models were built to assess the effect of HLA-B27 and gender on age at onset of axial symptoms. Results: Of 2579 axSpA patients, 92% had an age at onset of axial symptoms <45 years, with only small variations across the geographical regions [Asia, n = 574 (94%); Europe and North America, n = 988 (92%); Latin America, n = 246 (89%); Middle East and North Africa, n = 771 (91%)]. Age at onset of axial symptoms was consistently lower in HLA-B27-positive patients {median 25 years [interquartile range (IQR) 19-32] vs 31 [IQR 22-39]} and male patients [median 25 years (IQR 19-33) vs 28 (IQR 21-37)], but in multivariable models an additional statistically significant effect of male gender independent of HLA-B27 was only found in Asia. Conclusion: Around the world, the great majority of axSpA patients had an age at onset of axial disease of <45 years, with HLA-B27 and male gender associated with earlier disease onset. Show less
Toupin-April, K.; Decary, S.; Wit, M. de; Meara, A.; Barton, J.L.; Fraenkel, L.; ... ; Tugwell, P. 2021
Objective: To gain consensus on the Outcome Measures in Rheumatology (OMERACT) core domain set for rheumatology trials of shared decision making (SDM) interventions.Methods: The process followed... Show moreObjective: To gain consensus on the Outcome Measures in Rheumatology (OMERACT) core domain set for rheumatology trials of shared decision making (SDM) interventions.Methods: The process followed the OMERACT Filter 2.1 methodology, and used consensus-building methods, with patients involved since the inception. After developing the draft core domain set in previous research, we conducted five steps: (i) improving the draft core domain set; (ii) developing and disseminating whiteboard videos to promote its understanding; (iii) conducting an electronic survey to gather feedback on the draft core domain set; (iv) finalizing the core domain set and developing summaries, a plenary session video and discussion boards to promote its understanding; and (v) conducting virtual workshops with voting to endorse the core domain set.Results: A total of 167 participants from 28 countries answered the survey (62% were patients/caregivers). Most participants rated domains as relevant (81%-95%) and clear (82%-93%). A total of 149 participants (n = 48 patients/caregivers, 101 clinicians/researchers) participated in virtual workshops and voted on the proposed core domain set which received endorsement by 95%. Endorsed domains are: 1-Knowledge of options, their potential benefits and harms; 2-Chosen option aligned with each patient's values and preferences; 3-Confidence in the chosen option; 4-Satisfaction with the decision-making process; 5-Adherence to the chosen option and 6-Potential negative consequences of the SDM intervention.Conclusion: We achieved consensus among an international group of stakeholders on the OMERACT core domain set for rheumatology trials of SDM interventions. Future research will develop the Core Outcome Measurement Set.Clinical significance: Prior to this study, there had been no consensus on the OMERACT core domain set for SDM interventions. The current study shows that the OMERACT core domain set achieved a high level of endorsement by key stakeholders, including patients/caregivers, clinicians and researchers.(c) 2021 Elsevier Inc. All rights reserved. Show less
The second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the... Show moreThe second trimester of human development is marked by asynchronous gonadal development hampering the isolation of homogenous populations of early and late fetal germ cells (FGCs). We evaluated the feasibility of using surface markers TNAP, PDPN, EPCAM and ITGA6 to isolate FGCs as well as human primordial germ cell-like cells (hPGCLCs) derived from embryonic stem cells (hESCs) from both sexes by fluorescence-activated cell sorting (FACS). Our results suggest that a combination of TNAP and PDPN was sufficient to separate populations of premeiotic FGCs and hPGCLCs in both sexes. This combination of antibodies also proved efficient in separating 'mitotic' from 'retinoic-acid responsive' female FGCs. Furthermore, we report that the differentiation efficiency of TNAP+PDPN+ hPGCLCs from hESCs was sex-independent, but the ability to propagate differed considerably between the sexes. In contrast to male, female hPGCLCs retained their characteristics and exhibited robust colony-forming ability when cultured for five days in medium containing LIF, forskolin and FGF2. We conclude that marked sex differences exist in the isolation and propagation of human FGCs and hPGCLCs. Our study provides novel insights relevant for the optimization of in vitro gametogenesis in humans. Show less
Mishra, S.; Taelman, J.; Popovic, M.; Tilleman, L.; Duthoo, E.; Jeught, M. van der; ... ; Heindryckx, B. 2021
STUDY QUESTIONWhat is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?SUMMARY ANSWERPOU5F1 is required for successful... Show moreSTUDY QUESTIONWhat is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model?SUMMARY ANSWERPOU5F1 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 ALREADYClustered 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, DURATIONThe 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, METHODSA 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 CHANCEGenomic 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 CAUTIONOne 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 FINDINGSComparison 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. Show less
Boel, A.; Navarro-Compan, V.; Landewe, R.; Heijde, D. van der 2021
Objectives: There are two different approaches to involve participants in consecutive rounds of a Delphi survey: (1) invitation to every round independent of response to the previous round ("all... Show moreObjectives: There are two different approaches to involve participants in consecutive rounds of a Delphi survey: (1) invitation to every round independent of response to the previous round ("all-rounds'') and (2) invitation only when responded to the previous round ("respondents-only''). This study aimed to investigate the effect of invitation approach on the response rate and final outcome of a Delphi survey.Study Design and Setting: Both experts (N = 188) and patients (N = 188) took part in a Delphi survey to update the core outcome set (COS) for axial spondyloarthritis. A study with 1:1 allocation to two experimental groups (ie, "all-rounds'' [N = 187] and "respondents-only'' [N = 189]) was built-in.Results: The overall response rate was lower in the "respondents-only group'' (46%) compared to the "all-rounds group'' (61%). All domains that were selected for inclusion in the COS by the "respondents-only group'' were also selected by the "all-rounds group.'' Additionally, the four most important domains were identical between groups after the final round, with only minor differences in the other domains.Conclusion: Inviting panel members who missed a round to a subsequent round will lead to a better representation of opinions of the originally invited panel and reduces the chance of false consensus, while it does not influence the final outcome of the Delphi. (C) 2020 The Authors. Published by Elsevier Inc. Show less
Boel, A.; Molto, A.; Heijde, D. van der; Ciurea, A.; Dougados, M.; Gensler, L.S.; ... ; Ramiro, S. 2019
