Search

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

STUDY QUESTION Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S) A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

Aims Patients with cardiac disease are considered high risk for poor outcomes following hospitalization with COVID-19. The primary aim of this study was to evaluate heterogeneity in associations between various heart disease subtypes and in-hospital mortality. Methods and results We used data from the CAPACITY-COVID registry and LEOSS study. Multivariable Poisson regression models were fitted to assess the association between different types of pre-existing heart disease and in-hospital mortality. A total of 16 511 patients with COVID-19 were included (21.1% aged 66–75 years; 40.2% female) and 31.5% had a history of heart disease. Patients with heart disease were older, predominantly male, and often had other comorbid conditions when compared with those without. Mortality was higher in patients with cardiac disease (29.7%; n = 1545 vs. 15.9%; n = 1797). However, following multivariable adjustment, this difference was not significant [adjusted risk ratio (aRR) 1.08, 95% confidence interval (CI) 1.02–1.15; P = 0.12 (corrected for multiple testing)]. Associations with in-hospital mortality by heart disease subtypes differed considerably, with the strongest association for heart failure (aRR 1.19, 95% CI 1.10–1.30; P Conclusion Considerable heterogeneity exists in the strength of association between heart disease subtypes and in-hospital mortality. Of all patients with heart disease, those with heart failure are at greatest risk of death when hospitalized with COVID-19. Serious cardiac complications are rare during hospitalization.

Study Objectives International guidelines recommend using benzodiazepine receptor agonists (BZRA) for maximally four weeks. Nevertheless, long-term use for chronic insomnia disorder remains a common practice. This study aimed to test the effectiveness of blended care for discontinuing long-term BZRA use in general practice. Methods A pragmatic cluster randomized controlled superiority trial compared blended care to usual care through urine toxicology screening. In the intervention, care by the general practitioner (GP) was complemented by an interactive e-learning program, based on cognitive behavioral therapy for insomnia. Adults using BZRA daily for minimally 6 months were eligible. Participants were clustered at the level of the GP surgery for allocation (1:1). Effectiveness was measured as the proportion of patients who had discontinued at one-year follow-up. Data analysis followed intention-to-treat principles. Results In total, 916 patients in 86 clusters, represented by 99 GPs, were randomized. Primary outcome data was obtained from 727 patients (79%). At one-year follow-up, 82 patients (18%) in blended care, compared to 91 patients (20%) in usual care, had discontinued. There was no statistically significant effect for the intervention (OR: 0.924; 95% CI: 0.60; 1.43). No adverse events were reported to the research team. Conclusions The findings did not support the superiority of blended care over usual care. Both strategies showed clinical effectiveness, with an average of 19% of patients having discontinued at one-year follow-up. Further research is important to study the effect of structurally implementing digital interventions in general practice. Clinical trial Big Bird trial; KCE-17016. This trial is registered at clinicaltrials.gov (NCT03937180).

Background Primary health care providers (PHCPs) are assumed to be at high risk of a COVID-19 infection, as they are exposed to patients with usually less personal protective equipment (PPE) than other frontline health care workers (HCWs). Nevertheless, current research efforts focussed on the assessment of COVID-19 seroprevalence rates in the general population or hospital HCWs. Objective We aimed to determine the seroprevalence in PHCPs during the second SARS-CoV-2 wave in Flanders (Belgium) and compared it to the seroprevalence in the general population. We also assessed risk factors, availability of PPE and attitudes towards the government guidelines over time. Methods A prospective cohort of PHCPs (n = 698), mainly general practitioners, was asked to complete a questionnaire and self-sample capillary blood by finger-pricking at five distinct points in time (June–December 2020). We analysed the dried blood spots for IgG antibodies using a Luminex multiplex immunoassay. Results The seroprevalence of PHCPs remained stable between June and September (4.6–5.0%), increased significantly from October to December (8.1–13.4%) and was significantly higher than the seroprevalence of the general population. The majority of PHCPs were concerned about becoming infected, had adequate PPE and showed increasing confidence in government guidelines. Conclusions The marked increase in seroprevalence during the second COVID-19 wave shows that PHCPs were more at risk during the second wave compared to the first wave in Flanders. This increase was only slightly higher in PHCPs than in the general population suggesting that the occupational health measures implemented provided sufficient protection when managing patients.

Study question How do patients perceive the pre-IVF treatment communication by the nursing and midwifery team and how do they evaluate implemented optimization? Summary answer Patient satisfaction with the pre-IVF treatment communication by the nursing and midwifery team improved from 86% to 98% after implementation of standardized written patient information. What is known already Nurses are the main point of contact for patients undergoing medically assisted reproduction techniques (Applegarth et al., 2008). Morris (2001) suggested that infertility nurses could more specifically be involved in informative counselling in anticipation of the fertility treatment. Specific training for nurses and midwives would be required in order to achieve that central role in effective fertility counselling (Applegarth et al., 2012). However, little has been published in peer reviewed literature on how to assess and improve these pre-IVF treatment information sessions. Study design, size, duration The pre-IVF communication by the nurse/midwife was assessed by female patients with a self-developed questionnaire (cohort 1). The nursing and midwifery team was subsequently informed about the results of the questionnaire and efforts were undertaken to optimise future patient communication. Four years later, a follow-up questionnaire assessed patient satisfaction about the intervention (cohort 2). Each cohort comprised fifty patients and nurses and midwives were blinded for patient participation to the study. Participants/materials, setting, methods The study was performed at an academic fertility centre and was approved by the Investigational Review Board. Data were collected with SurveyMonkey (pre intervention) and REDCap (post intervention). A descriptive analysis of the cohorts and patient feedback was performed. The intervention consisted of (1) the optimization of patient information documents if needed (2) providing a training to the nursing and midwifery staff, and (3) a follow-up questionnaire in a second cohort of female fertility patients. Main results and the role of chance The first questionnaire revealed that overall, patients were satisfied with the informative counselling session in anticipation of the IVF treatment. However, some patients indicated that they received a lot of information at once and that specific information that was of relevance for the further treatment (e.g. oocyte pick-up) was sometimes missed. Also, the data showed that not all patients received the same information. Therefore, all information that patients needed during an IVF treatment was gathered into a patient binder. It was supplemented with a timeline of the whole IVF trajectory, QR codes to movies explaining the administration of medication, and answers to common patients’ questions. It was possible to individualize the content of the binder per patient. Ideally, patients received this binder before the informative counselling session so they could read the content in advance. The binder was developed by a member of the nursing and midwifery team and was reviewed by the medical, laboratory, and administrative staff. Then, the nursing and midwifery team was trained on the use of the patient binder. The follow-up questionnaire revealed that patient satisfaction with the pre-IVF treatment counselling was 98% after the optimization of patient documentation (compared to 86% before the optimization). Limitations, reasons for caution Eligible patients were recruited by the treating physician. As no records were kept of the number of solicited patients for this study, no assessment of the response rate is possible. The cohorts were self-selected and limited in size and could therefore not reflect the general patient population. Wider implications of the findings A patient questionnaire and follow-up is a useful tool for a centre specific assessment and improvement of pre-IVF nurse/midwife communication with patients. Clinics could make efforts to invest in complete and written information and ask colleagues of the nursing and midwifery team to be involved in its creation. Trial registration number NCT04420169

Study question Could collection temperature and spindle transfer (ST) potentially improve development of embryos derived from in vitro matured (IVM) ovarian tissue oocytes (OTO) of transgender men? Summary answer Spindle transfer, but not collection temperature, significantly improved embryo development of OTO-IVM oocytes from transgender men. What is known already For transgender men, the fertility preservation strategy of ovarian stimulation may interfere with the desired masculine characteristics and enhance gender dysphoria. Alternatively, ovarian tissue oocytes collected ex vivo could serve as potential gametes, not requiring ovarian stimulation. Oocytes can be collected during gender affirming surgery, matured, and vitrified. Ovarian tissue oocyte in vitro maturation (OTO-IVM) has successfully been used for cancer patients, as live births have been reported. OTO-IVM in transgender men demonstrated sufficient maturation rates and survival following vitrification. Nevertheless, a decreased fertilization potential of these oocytes and severely compromised embryonic development have been observed. Study design, size, duration Patients between 18-24 years were recruited for this study from November 2020 to September 2021. Ovaries from 14 transgender men were collected in either cold (4oC, OTO-Cold) or warm (37oC, OTO-Warm) collection medium, to verify the best collection method. Following ovarian manipulation, cumulus oocyte complexes (COCs) were harvested from spent medium and underwent maturation for 48hrs. ST was performed to overcome inferior fertilization and embryonic development. Participants/materials, setting, methods Injected IVM oocytes underwent calcium imaging or were monitored for embryonic developmental potential. In vitro matured GV (germinal vesicle), MI (metaphase I) and in vivo matured oocytes with clusters of smooth endoplasmic reticulum (SERa) served as controls and cytoplasmic recipients for ST. OTO-IVM or control oocytes were used as spindle donors (ST-OTO or Control-ST respectively). Genetic analysis was performed to detect chromosomal abnormalities in embryos from all groups. Main results and the role of chance In total, we collected 252 OTO-Cold and 230 OTO-Warm oocytes, showing similar maturation rates (53%). For calcium imaging, 39 control, 33 OTO-cold and 31 OTO-warm oocytes were analysed, determining the product of amplitude per frequency, in arbitrary units (AU). The average value for control oocytes was 2.30AU, significantly higher than OTO-Cold (1.47AU, p=0.046) and OTO-Warm oocytes (1.03AU, p=0.036). Calcium release was similar between OTO-Cold and OTO-Warm oocytes. Following ICSI, 19/47 OTO-Cold and 24/48 OTO-Warm oocytes normally fertilized, significantly lower than the control group (42/52) (p Limitations, reasons for caution A major limitation of our study is the lack of ovaries from cis women. Control oocytes used in this study originate from infertility patients that underwent ovarian stimulation. High abnormality rate in ST-OTO embryos might be concerning for the safety of ST, but the number of embryos analysed is limited. Wider implications of the findings Our data indicate that OTO-IVM oocytes from transgender men display poor cytoplasmic quality, demonstrated by embryonic arrest and calcium imaging. ST was able to overcome poor embryo development, and it could be of interest to use freshly donated oocytes as cytoplasmic recipients for this. Trial registration number Not applicable

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation Flanders Introduction Myocarditis is an inflammatory disease of the heart with viral infections being the most common aetiology. Often, myocardial fibrosis (MF) is observed during the resolution of myocarditis. MF is a strong independent predictor for outcome in patients with myocarditis. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. Purpose The primary aim of this study was to characterise the natural history of coxsackievirus B3 (CVB3)-induced myocarditis in C57BL/6J mice, with emphasis on fibrogenesis and its electrical impact. Secondary aims were 1) to study the impact of inoculation dose and sex on the course of disease, and 2) to develop an in-house histopathological scoring system for inflammation and fibrosis. Methods C57BL/6J mice (12 weeks old; n=154) received intraperitoneal injection with CVB3 (Nancy strain). Male mice received 5 x 10^5 (regular dose)(RD) or 5 x 10^6 (high dose) plaque-forming units, whereas female mice received the RD only. Mice injected with PBS served as control groups. Animals were sacrificed 7, 14, 28, 56 and 77 days after CVB3 or PBS injection. Inflammation (haematoxylin-eosin) and fibrosis (Picrosirius red (PSR)) were evaluated histologically. Arrhythmogenicity was assessed through right ventricular programmed stimulation. Results CVB3 animals sacrificed or with spontaneous death within 14 days after inoculation showed cardiac infiltration in all (100%) and necrosis in 88% (35/40). In male CVB3-infected mice, premature mortality was ±20% in both regular and high dose groups, occurring between 9 and 23 days post inoculation. No mortality was observed in the female group (p Conclusion We report on the characterisation of fibrotic remodelling in a CVB3-induced myocarditis mouse model. Fibrotic remodelling was seen in a majority of mice after 8 to 11 weeks. It was associated with a numerical (but not significant) increased arrhythmogenicity, requiring further exploration given the clinical significance of fibrotic remodelling. Further research on the molecular pathways and cellular interactions triggered by myocarditis is ongoing.

Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G>C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p > 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (>18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

Study question Can oocytes isolated from transgender men after oophorectomy support embryonic development? Summary answer Embryo developmental arrest at 4–8cell stage (day 3 embryos) indicates poor quality of in vitro matured oocytes from transgender men. What is known already Gender affirming surgery for transgender men leads to permanent infertility, as it involves bilateral oophorectomy. Current approaches for fertility preservation, such as oocyte freezing following ovarian hyperstimulation, may interfere with the wanted masculine characteristics and enhance gender dysphoria. In vitro matured oocytes (IVM) isolated following oophorectomy have been proposed as a source of potential gametes to ensure fertility preservation for transgender men with a child wish. From previous studies, it has been shown that these oocytes are able to undergo maturation and display normal spindles, but their competence to be fertilized and support embryonic development has not been addressed yet. Study design, size, duration We evaluated the quality of in vitro matured oocytes isolated from ovaries of transgender men by applying calcium imaging and monitoring fertilization and embryonic development following intracytoplasmic sperm injection (ICSI). Ovaries were collected in cold (4oC) or warm (37oC) medium, to investigate the best collection procedure. So far, results from four transgender men have been included. Participants/materials, setting, methods: Ovaries from four transgender men undergoing testosterone treatment were collected after oophorectomy in cold or warm medium. Cumulus oocyte complexes (COCs) were isolated and cultured in maturation medium for 48hrs. Mature oocytes were injected with donated sperm and assessed either by calcium imaging, measuring the total calcium release following injection, or following embryonic development. Donated in vitro matured oocytes, germinal vesicle(GV) or metaphase I(MI) origin, from other patients undergoing IVF treatment were used as controls. Main results and the role of chance In total, 179 COCs were collected from ovaries (n = 8) of four transgender men. From the COCs collected in warm medium, 73/105(69%) survived and 33/73(45%) reached metaphase II (MII). Of 21 MII injected with sperm, 13/21(62%) fertilized, 9/21(43%) formed 2 pronuclei (PN), 8/9(89%) reached the 2-cell stage, 3/9(33%) reached 4–8cell stage but arrested. From 74 COCs isolated in cold medium, 57/74(77%) survived and 28/57(49%) matured. Of the 11 MII injected with sperm, 7/11(64%) fertilized, 6/11(54%) formed 2PN, 6/6(100%) reached the 2-cell stage, 4/6(67%) reached 4–8cell but arrested. In the control group, 10/13 oocytes injected with the same sperm sample, were normally fertilized (77%), 8/10(80%) reached the 2-cell stage, 7/10(70%) reached the 4–8cell stage and 4/10(40%) became blastocysts. From the warm, cold and control conditions, respectively 12,14 and 17 MII oocytes were used for calcium imaging. The product of amplitude and frequency of calcium peaks, representing total calcium release, was calculated. Oocytes showed an average release of 0.66AU and 1.69AU for the warm and cold condition, respectively. The average value for control oocytes was 2.19AU. Limitations, reasons for caution One major limitation of our study is the lack of ovaries from cis women as control group. Our control oocytes originated from women undergoing IVF treatment and have undergone ovarian stimulation. Furthermore, the number of oocytes analysed and number of patients per group was limited and is being increased. Wider implications of the findings: Our data indicate that in vitro matured oocytes from transgender men ovaries display poor quality, as demonstrated by the poor embryonic development. In the future, we will apply nuclear transfer technology as a mean to overcome embryonic developmental arrest in this group of oocytes. Trial registration number Not applicable

Background The effect of adherence to cardiac rehabilitation (CR) on outcome is not clear. Therefore, we aimed to assess the impact of drop-out for non-medical reasons of CR on event-free survival in coronary artery disease (CAD). Methods A total of 876 patients who attended CR after acute coronary syndrome (ACS), percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) were included. Drop-out was defined as attending ≤50% of the training sessions. A combined endpoint of all-cause mortality and rehospitalization for a cardiovascular event was used to specify event-free survival. Differences in clinical characteristics were assessed and parameters with p Results A total of 15% died or had a cardiovascular event during a median follow-up period of 33 months (interquartile range 24, 51). Overall, 17% dropped out before finishing half of the program. Patients who withdrew prematurely had a risk twice as high for a cardiovascular event or death (hazard ratio 1.92, 95% confidence interval 1.28–2.90) than those who attended more than half of the sessions. Both ACS (2.36, 1.47–3.58) and PCI (2.20, 1.22–3.96), as primary indicators for CR, were associated with an adverse outcome and also a prior history of chronic heart failure (CHF) remained negatively associated with event-free survival (3.67, 1.24–10.91). Finally, the presence of hyperlipidemia was independently related to a worse outcome (1.48, 1.02–2.16). Conclusions Drop-out for non-medical reasons was independently associated with a negative outcome in CAD. Therefore, underlying factors for drop-out should gain more attention in future research and should be taken into account when organizing CR.

Background Despite improvements in medical and surgical therapy, infective endocarditis (IE) remains a deadly disease. Echocardiography is the first-line diagnostic tool. However, data regarding its role in the prognostic assessment of in-hospital clinical outcome of IE are scarce. Purpose We sought to assess the role of echocardiography to predict the in-hospital outcome in a large cohort of patients diagnosed with definite IE and its association with clinical presentation and microorganisms. Methods We retrospectively included patients from two centers between 2006 and 2018. Transthoracic and transesophageal echocardiography were performed in all patients. The clinical endpoints were in-hospital death, embolic events (cerebrovascular and non-cerebrovascular), shock (septic shock and cardiogenic shock) and cardiac surgery. Results 183 patients with definite IE (age 68.9 ± 14.2 years old, 68.9% male) were evaluated. Ninety three (50.8%) patients had aortic valve IE and 81 (44.3%) patients presented with mitral valve IE. Twenty three patients had multivalvular IE. The in-hospital mortality rate was 22.4%. Sixty patients (32.8%) had embolic events and 42 (23%) patients developed shock during hospitalization. Surgery was performed in 103 (56.3%) patients. Mitral valve IE on echocardiography was an independent predictor of in-hospital mortality (p = 0.038, OR 0.38, 95% CI 0.15 – 0.94) and aortic valve IE on echocardiography was an independent predictor of embolic events (p = 0.018, OR 0.36, 95% 0.16-0.84). The presence of a new cardiac murmur upon admission was predictive for the need of cardiac surgery (p = 0.042, OR 0.51, 95% CI 0.22- 1.09) and correlated with the severity of valvular regurgitation identified by echocardiography (p = 0.024). Methicillin resistant Staphylococcus aureus (MRSA) as the causative microorganism was an independent predictor for in - hospital mortality and for the development of shock during hospitalization (p = 0.010, OR 0.13 95% CI 0.30 - 0.62 and p = 0.027, OR 6.11, 95% CI 1.22 – 30.37, respectively). No correlation was found between MRSA and echocardiographic parameters. Conclusion Mitral valve IE was an independent predictor of in - hospital mortality. Furthermore, aortic valve IE was an independent predictor of embolic events. The presence of a new cardiac murmur was predictive for the need of cardiac surgery and correlated with the severity of valvular regurgitation by echocardiography. Our findings suggest that a thorough physical examination upon admission is required in combination with a comprehensive echocardiographic exam for early identification of patients with IE at high - risk for in-hospital death and complications.

Background The emergence of non-vitamin K antagonist oral anticoagulants (NOAC) has caused a paradigm shift in anticoagulation therapy, with NOAC being increasingly used compared to vitamin K antagonists. Despite the numerous RCTs with NOAC, further follow-up on how this high risk new drug class is used in real-world clinical practice is warranted. Purpose This study aimed to describe NOAC use in a primary care sample of long-term NOAC users, and investigated (i) adherence, (ii) patients' perceptions, (iii) drug interactions and (iv) appropriateness of dosing. Methods A cross-sectional observational study was conducted in 158 community pharmacies. Participants were home-dwelling adults treated with a NOAC for at least one year. They completed a questionnaire collecting data on socio-demographics, clinical characteristics, current medication use, self-reported adherence to NOAC (via Medication Adherence Report Scale, MARS), and beliefs and perceptions about NOAC (via Beliefs about Medicines Questionnaire, BMQ). Adherence to NOAC (self-reported (MARS) and calculated using pharmacy dispensing data (Medication Possession Ratio (MPR)); patients' beliefs and perceptions about NOAC (BMQ); and prevalence of drug interactions and inappropriate dosing (using the recommendations in the summary of product characteristics (SmPC) and the 2018 European Heart Rhythm Association (EHRA) Practical Guide) were assessed. Results Participants (n=766) had a mean age of 76.2±8.8 years, several co-morbidities (median of 5 (IQR 4–6)) and high thromboembolic risk (median CHA2DS2-VASc score of 4 (IQR 3–4)). The majority (93.5%) used NOAC for non-valvular atrial fibrillation, while the other 6.5% used it for the prevention of recurrent deep vein thrombosis or pulmonary embolism. Forty point five percent of the study sample used rivaroxaban, 36.2% apixaban, 21.1% dabigatran and 2.2% edoxaban. About 85% was adherent according to MPR (MPR ≥80%) and self-reported adherence was also high (mean MARS score 24.6±1.0). Two-thirds reported at least one adverse event of the NOAC, with easy bruising/bleeding being most prevalent (40.2% of patients). BMQ showed that 91.3% of patients favoured the “necessity” over the “concerns” of NOAC use. Thirty-one percent of patients had ≥1 drug interaction(s) with NOAC; amiodarone (10.4% of patients), antiplatelet agents (9.7%) and NSAIDs (9.0%) were the most common interacting drugs. NOAC dosing was inappropriate in 15.9% of patients according to SmPC and in 23.1% according to EHRA. Intriguingly, underdosing was more common using the SmPC as reference, while overdosing was more common using the EHRA guideline. Conclusions This real-world analysis of NOAC use revealed high adherence and necessity beliefs, drug interactions in 30% of patients, and suboptimal dosing in about one fifth of patients. These findings can inform the design of targeted community pharmacist interventions to improve quality of NOAC use.

BACKGROUND: To simplify the assessment of the recording and control of coronary heart disease risk factors in different countries and regions. DESIGN: The SUrvey of Risk Factors (SURF) is an international clinical audit. METHODS: Data on consecutive patients with established coronary heart disease from countries in Europe, Asia and the Middle East were collected on a one-page collection sheet or electronically during routine clinic visits. Information on demographics, diagnostic category, risk factors, physical and laboratory measurements, and medications were included and key variables summarized in a Cardiovascular Health Index Score. RESULTS: Coronary heart disease patients (N = 10,186; 29% women) were enrolled from 79 centres in 11 countries. Recording of risk factors varied considerably: smoking was recorded in over 98% of subjects, while about 20% lacked data on laboratory measurements relevant to cardiovascular disease risk. Sixteen per cent of participants reported smoking, 29% were obese, and 46% had abdominal obesity. Sixty per cent of participants had blood pressure

STUDY QUESTION: Do overweight women with polycystic ovary syndrome (PCOS) have a higher risk of perinatal complications than normal weight women with PCOS? SUMMARY ANSWER: Overweight women with PCOS with an ongoing singleton pregnancy have an increased risk of preterm birth as well as an increased risk of giving birth to a baby with a higher birthweight than normal weight women with PCOS. WHAT IS KNOWN ALREADY: There is evidence that overweight (BMI > 25 kg/m²) has a negative influence on the prevalence of gestational diabetes mellitus and fetal macrosomia in women with PCOS. STUDY DESIGN, SIZE, DURATION: We set up a retrospective comparative cohort study of 93 overweight (BMI ≥ 25 kg/m²) and 107 normal weight (BMI < 25 kg/m²) women with PCOS who were scheduled for fertility treatment between January 2000 and December 2009 and achieved a pregnancy as a result of a treatment cycle, or spontaneously before or between treatment cycles. PARTICIPANTS/MATERIALS, SETTING, METHODS: All data (patient characteristics, medical information, pregnancy, delivery and neonatal outcome) were retrieved from patient medical files. All pregnancy, delivery and neonatal outcome parameters were adjusted for age and pre-pregnancy smoking behaviour. The neonatal outcome parameters were additionally adjusted for gestational age. MAIN RESULTS AND THE ROLE OF CHANCE: The median BMI in the overweight and normal weight women was, respectively, 30.8 kg/m² [interquartile quartile range (IQR) 5.8] and 20.9 kg/m² (IQR 2.3) (P < 0.001). Baseline characteristics did not differ between groups, except for free testosterone and fasting insulin levels, which were higher, and sex hormone-binding globulin, which was lower, in overweight versus normal weight women (all P < 0.001). The time-to-pregnancy was significantly higher in the overweight group (P = 0.01). Multivariate analyses of the ongoing singleton pregnancies showed significantly more preterm births in overweight (10/61) versus normal weight (2/71) women [adjusted odds ratio 0.1, 95% confidence interval (CI) 0-0.6, P = 0.01]. The mean birthweight of newborns was significantly higher in overweight (3386 ± 663 g) than in normal weight (3251 ± 528 g) women (adjusted mean difference 259.4, 95% CI 83.4-435.4, P = 0.004). LIMITATIONS, REASON FOR CAUTION: Our results only represent the pregnancy, delivery and neonatal outcome of ongoing singleton pregnancies. The rather small sample size and observational nature of the study are further limitations. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest the importance of pre-pregnancy weight loss in overweight women with PCOS in order to reduce the risk of adverse perinatal outcomes. STUDY FUNDING/COMPETING INTERESTS: Veerle De Frene is holder of a Special PhD Fellowship by the Flemish Foundation for Scientific Research (FWO-Vlaanderen). Petra De Sutter is holder of a fundamental clinical research mandate by the Flemish Foundation for Scientific Research (FWO-Vlaanderen). There are no competing interests.

Study question: What meanings do lesbian couples construct regarding their sperm donor? Summary answer: Forsome parents, the donor was increasingly presented as a person, whereas for other parents, the donor was seen as an instrument from the moment they received the sperm donation. What is known already: Few studies specifically focus on how lesbian couples deal with the issue of third-party anonymous gamete donation. It is often assumed that they have fewer difficulties than heterosexual couples with the involvement of a male procreator, since their status as a donor conception family is ‘socially visible’ and there is no social father who fears exclusion. Study design, size, duration: Semi-structured interviews were conducted with 10 lesbian couples (20 participants), recruited via the Ghent University Hospital. All couples had at least one child, conceived through anonymous donor insemination, between 7 and 10 years old. Participants/materials, setting, methods: Within the data corpus, a particular data set was analyzed where couples referred to their donor and his position in their family. Step-by-step inductive thematic analysiswas performed resulting in themes that are grounded in the data. All phases of the analysis were followed by team discussion. Main results and the role of chance: This study reveals different donor constructs, indicating different ways of dealing with the third-party involvement in the family. Some parents diminish the role of the donor throughout family life and continue to present him as an instrument: something they needed in order to become parents. Others showan increasing interest in the donor as the children mature, which results in a more personalized account of the donor. Limitations, reasons for caution: In our qualitative cross-sectional study, we collected retrospectively constructed stories. Longitudinal qualitative and quantitative research is required to allow for an extrapolation of the conclusions made. Wider implications of the findings: This study shows how the concept of the donor is constructed within lesbian families and howit is challenged by the child’s developing personality and features.Whencounseling prospective parents, it could therefore be useful to discuss the concept of the anonymous donor beyond the conception phase. Study funding/competing interest(s): The project was funded by the Research Fund of Ghent University, Belgium. There are no competing interests.