Association between the length of in vitro embryo culture, mode of ART, and the initial endogenous hCG rise in ongoing singleton pregnancies.

STUDY QUESTION: Is there an association between the length of in vitro culture, mode of ART and the initial endogenous hCG rise, in cycles with a foetal heartbeat after single embryo transfer (ET) and implantation? SUMMARY ANSWER: Both the length of in vitro culture and the mode of ART have an impact on the initial endogenous rise in hCG in singleton pregnancies. WHAT IS KNOWN ALREADY: Different factors have been identified to alter the kinetics of hCG in pregnancies. Current studies show conflicting results regarding the kinetics of hCG after different types of ART (fresh vs frozen ET (FET)), the inclusion or not of preimplantation genetic testing (PGT), and the length of time in in vitro culture. STUDY DESIGN, SIZE, DURATION: This was a multicentre cohort study, using prospectively collected data derived from 4938 women (5524 treatment cycles) undergoing IUI (cycles, n = 608) or ART (cycles, n = 4916) treatments, resulting a in singleton ongoing pregnancy verified by first-trimester ultrasound scan. Data were collected from the Danish Medical Data Centre, used by the three participating Danish public fertility clinics at Copenhagen University hospitals: Herlev Hospital, Hvidovre Hospital, and Rigshospitalet, from January 2014 to December 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The fresh ET cycles included cleavage-stage (2 or 3 days in vitro) and blastocyst (5 days in vitro) transfers. FET cycles included cleavage-stage (3 days in vitro before cryopreservation) or blastocyst (5 or 6 days in vitro before cryopreservation) transfers. The IUI cycles represented no time in vitro. To attain a comparable interval for serum-hCG (s-hCG), the ovulation induction time was identical: 35-37 h before oocyte retrieval or IUI. The conception day was considered as: the insemination day for pregnancies conceived after IUI, the oocyte retrieval day for fresh ET, or the transfer day minus 3 or 5 as appropriate for FET of Day 3 or 5 embryos. Multiple linear regression analysis was used, including days post-conception for the hCG measurement as a covariate, and was adjusted for the women's age, the cause of infertility, and the centre. For FET, a sensitivity analysis was used to adjust for endometrial preparation. MAIN RESULTS AND THE ROLE OF CHANCE: The study totally includes 5524 cycles: 2395 FET cycles, 2521 fresh ET cycles, and 608 IUI cycles. Regarding the length of in vitro culture, with IUI as reference (for no time in in vitro culture), we found a significantly lower s-hCG in pregnancies achieved after fresh ET (cleavage-stage ET or blastocyst transfer). S-hCG was 18% (95% CI: 13-23%, P < 0.001) lower after fresh cleavage-stage ET, and 23% (95% CI: 18-28%, P < 0.001) lower after fresh blastocyst transfer compared to IUI. In FET cycles, s-hCG was significantly higher after blastocyst transfers compared to cleavage-stage FET, respectively, 26% (95% CI: 13-40%, P < 0.001) higher when cryopreserved on in vitro Day 5, and 14% (95% CI: 2-26%, P = 0.02) higher when cryopreserved on in vitro Day 6 as compared to Day 3. Regarding the ART treatment type, s-hCG after FET blastocyst transfer (Day 5 blastocysts) cycles was significantly higher, 33% (95% CI: 27-45%, P < 0.001), compared to fresh ET (Day 5 blastocyst), while there was no difference between cleavage-stage FET (Days 2 + 3) and fresh ET (Days 2 + 3). S-hCG was 12% (95% CI: 4-19%, 0.005) lower in PGT FET (Day 5 blastocysts) cycles as compared to FET cycles without PGT (Day 5 blastocysts). LIMITATIONS, REASONS FOR CAUTION: The retrospective design is a limitation which introduces the risk of possible bias and confounders such as embryo score, parity, and ovarian stimulation. WIDER IMPLICATIONS OF THE FINDINGS: This study elucidates how practices in medically assisted reproduction treatment are associated with the hCG kinetics, underlining a potential impact of in vitro culture length and mode of ART on the very early embryo development and implantation. The study provides clinicians knowledge that the type of ART used may be relevant to take into account when evaluating s-hCG for the prognosis of the pregnancy. STUDY FUNDING/COMPETING INTEREST(S): No funding was received for this study. AP has received consulting fees, research grants, or honoraria from the following companies: Preglem, Novo Nordisk, Ferring Pharmaceuticals, Gedeon Richter, Cryos, Merck A/S, and Organon. AZ has received grants and honoraria from Gedeon Richter. NLF has received grants from Gedeon Richter, Merck A/S, and Cryos. MLG has received honoraria fees or research grants from Gedeon Richter, Merck A/S, and Cooper Surgical. CB has received honoraria from Merck A/S. MB has received research grants and honoraria from IBSA. MPR, KM, and PVS all report no conflicts of interest. TRIAL REGISTRATION NUMBER: The study was registered and approved by the Danish Protection Agency, Capital Region, Denmark (Journal-nr.: 21019857). No approval was required from the regional ethics committee according to Danish law.

Androgen and inhibin B levels during ovarian stimulation before and after 8 weeks of low-dose hCG priming in women with low ovarian reserve.

STUDY QUESTION: Does 8 weeks of daily low-dose hCG administration affect androgen or inhibin B levels in serum and/or follicular fluid (FF) during the subsequent IVF/ICSI cycle in women with low ovarian reserve? SUMMARY ANSWER: Androgen levels in serum and FF, and inhibin B levels in serum, decreased following 8 weeks of hCG administration. WHAT IS KNOWN ALREADY: Recently, we showed that 8 weeks of low-dose hCG priming, in between two IVF/ICSI treatments in women with poor ovarian responder (anti-Müllerian hormone (AMH) <6.29 pmol/l), resulted in more follicles of 2-5 mm and less of 6-10-mm diameter at the start of stimulation and more retrieved oocytes at oocyte retrieval. The duration of stimulation and total FSH consumption was increased in the IVF/ICSI cycle after priming. Hypothetically, hCG priming stimulates intraovarian androgen synthesis causing upregulation of FSH receptors (FSHR) on granulosa cells. It was therefore unexpected that antral follicles were smaller and the stimulation time longer after hCG priming. This might indicate a different mechanism of action than previously suggested. STUDY DESIGN, SIZE, DURATION: Blood samples were drawn on stimulation day 1, stimulation days 5-6, trigger day, day of oocyte retrieval, and oocyte retrieval + 5 days in the IVF/ICSI cycles before and after hCG priming (the control and study cycles, respectively). FF was collected from the first aspirated follicle on both sides during oocyte retrieval in both cycles. The study was conducted as a prospective, paired, non-blinded, single-center study conducted between January 2021 and July 2021 at a tertiary care center. The 20 participants underwent two identical IVF/ICSI treatments: a control cycle including elective freezing of all blastocysts and a study cycle with fresh blastocyst transfer. The control and study cycles were separated by 8 weeks (two menstrual cycles) of hCG priming by daily injections of 260 IU recombinant hCG. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women aged 18-40 years with cycle lengths of 23-35 days and AMH <6.29 pmol/l were included. Control and study IVF/ICSI cycles were performed in a fixed GnRH-antagonist protocol. MAIN RESULTS AND THE ROLE OF CHANCE: Inhibin B was lower on stimulation day 1 after hCG priming (P = 0.05). Dehydroepiandrosterone sulfate (DHEAS) was significantly lower on stimulation day 1 (P = 0.03), and DHEAS and androstenedione were significantly lower on stimulation days 5-6 after priming (P = 0.02 and P = 0.02) The testosterone level in FF was significantly lower in the study cycle (P = 0.008), while the concentrations of inhibin B and androstenedione in the FF did not differ between the study and control cycles. A lower serum inhibin B in the study cycle corresponds with the antral follicles being significantly smaller after priming, and this probably led to a longer stimulation time in the study cycle. This contradicts the theory that hCG priming increases the intraovarian androgen level, which in turn causes more FSHR on developing (antral up to preovulatory) follicles. However, based on this study, we cannot rule out that an increased intra-follicular androgen level was present at initiation of the ovarian stimulation, without elevating the androgen level in serum and that an increased androgen level may have rescued some small antral follicles that would have otherwise undergone atresia by the end of the previous menstrual cycle. We retrieved significantly more oocytes in the Study cycle, and the production of estradiol per follicle ≥10-mm diameter on trigger day was comparable in the study and control cycles, suggesting that the rescued follicles were competent in terms of producing oocytes and steroid hormones. LIMITATIONS, REASONS FOR CAUTION: The sample size was small, and the study was not randomized. Our study design did not allow for the measurement and comparison of androgen levels or FSHR expression in small antral follicles before and immediately after the hCG-priming period. WIDER IMPLICATIONS OF THE FINDINGS: The results make us question the mechanism of action behind hCG priming prior to IVF. It is important to design a study with the puncture of small antral follicles before and immediately after priming to investigate the proposed hypothesis. Improved cycle outcomes, i.e. more retrieved oocytes, must be confirmed in a larger, preferably randomized study. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by an unrestricted grant from Gedeon Richter awarded to the institution. A.P. reports personal consulting fees from PregLem SA, Novo Nordisk A/S, Ferring Pharmaceuticals A/S, Gedeon Richter Nordics AB, Cryos International, and Merck A/S outside the submitted work and payment or honoraria for lectures from Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, Merck A/S, and Theramex and Organon & Co and payment for participation in an advisory board for Preglem. Grants to the institution have been provided by Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, and Merck A/S, and equipment and travel support has been given to the institution by Gedeon Richter Nordics AB. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT04643925.

Eight weeks of androgen priming by daily low-dose hCG injections before ICSI treatment in women with low ovarian reserve.

STUDY QUESTION: Does 8 weeks of continuous low-dose hCG administration increase the proportion of antral follicles that reach the preovulatory state during ovarian stimulation (OS) in women with low ovarian reserve? SUMMARY ANSWER: The proportion of antral follicles (2-10 mm) that reached the preovulatory state did not increase. WHAT IS KNOWN ALREADY: The administration of androgens prior to OS might upregulate FSH receptor (FSHR) expression on granulosa cells, making follicles more responsive to exogenous FSH stimulation during OS. LH and hCG stimulate the local follicular androgen synthesis in theca cells and may be used as an endogenous androgen priming method. Exogenous priming by testosterone and dehydroepiandrosterone (DHEA) have been shown to increase the number of retrieved oocytes and live birth rate but the studies are small, and their use is associated with side effects. STUDY DESIGN, SIZE, DURATION: A prospective, paired, non-blinded single-center study including 20 women serving as their own controls conducted between January 2021 and July 2021 at The University Hospital Copenhagen Rigshospitalet, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants underwent two identical consecutive IVF/ICSI treatments, a Control cycle and a Study cycle, separated by ∼8 weeks (two menstrual cycles) of daily injections of 260 IU recombinant hCG (rhCG). A freeze-all strategy was applied in the Control cycle. Both IVF/ICSI cycles were performed in a fixed GnRH antagonist protocol using a daily dose of 300 IU recombinant FSH (rFSH) and GnRH antagonist 0.25 mg from stimulation days 5-6. MAIN RESULTS AND THE ROLE OF CHANCE: Follicular output rate, defined as the number of follicles >16 mm on hCG trigger day divided by the antral follicle count (2-10 mm) at baseline, did not increase after 8 weeks of hCG priming (P = 0.8). The mean number of oocytes retrieved was significantly higher after the hCG priming being 4.7 (2.8) vs 3.2 (1.7) in the Study and Control cycle, respectively (P = 0.01). The duration of stimulation was longer in the Study versus the Control cycle (P = 0.05), despite the use of identical hCG trigger criterion and similar diameters of the three biggest follicles on hCG trigger day in the two cycles (P = 0.9). LIMITATIONS, REASONS FOR CAUTION: The sample size was small, and the number of oocytes retrieved was not the primary endpoint. Larger studies are needed to confirm this finding. WIDER IMPLICATIONS OF THE FINDINGS: Long-term, low-dose rhCG administration may increase the number of oocytes retrieved during IVF/ICSI in women with low ovarian reserve, but more research is needed before firm conclusions can be drawn. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by an unrestricted grant from Gedeon Richter. A.P. reports personal consulting fees from PregLem SA, Novo Nordisk A/S, Ferring Pharmaceuticals A/S, Gedeon Richter Nordics AB, Cryos International, and Merck A/S outside the submitted work and payment or honoraria for lectures from Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, Merck A/S, and Theramex and Organon & Co. Grants to the institution have been provided by Gedeon Richter Nordics AB, Ferring Pharmaceuticals A/S, and Merck A/S and receipt of equipment by the institution from Gedeon Richter Nordics AB is reported. The remaining authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT04643925.

Patient-reported outcome measures for physical function in cancer patients: content comparison of the EORTC CAT Core, EORTC QLQ-C30, SF-36, FACT-G, and PROMIS measures using the International Classification of Functioning, Disability and Health.

BACKGROUND: Patient-reported physical function (PF) is a key endpoint in cancer clinical trials. Using complex statistical methods, common metrics have been developed to compare scores from different patient-reported outcome (PRO) measures, but such methods do not account for possible differences in questionnaire content. Therefore, the aim of our study was a content comparison of frequently used PRO measures for PF in cancer patients. METHODS: Relying on the framework of the International Classification of Functioning, Disability and Health (ICF) we categorized the item content of the physical domains of the following measures: EORTC CAT Core, EORTC QLQ-C30, SF-36, PROMIS Cancer Item Bank for Physical Function, PROMIS Short Form for Physical Function 20a, and the FACT-G. Item content was linked to ICF categories by two independent reviewers. RESULTS: The 118 items investigated were assigned to 3 components ('d - Activities and Participation', 'b - Body Functions', and 'e - Environmental Factors') and 11 first-level ICF categories. All PF items of the EORTC measures but one were assigned to the first-level ICF categories 'd4 - Mobility' and 'd5 - Self-care', all within the component 'd - Activities and Participation'. The SF-36 additionally included item content related to 'd9 - Community, social and civic life' and the PROMIS Short Form for Physical Function 20a also included content related to 'd6 - domestic life'. The PROMIS Cancer Item Bank (v1.1) covered, in addition, two first-level categories within the component 'b - Body Functions'. The FACT-G Physical Well-being scale was found to be the most diverse scale with item content partly not covered by the ICF framework. DISCUSSION: Our results provide information about conceptual differences between common PRO measures for the assessment of PF in cancer patients. Our results complement quantitative information on psychometric characteristics of these measures and provide a better understanding of the possibilities of establishing common metrics.

Development and psychometric evaluation of item banks for memory and attention - supplements to the EORTC CAT Core instrument.

BACKGROUND: Cancer patients may experience a decrease in cognitive functioning before, during and after cancer treatment. So far, the Quality of Life Group of the European Organisation for Research and Treatment of Cancer (EORTC QLG) developed an item bank to assess self-reported memory and attention within a single, cognitive functioning scale (CF) using computerized adaptive testing (EORTC CAT Core CF item bank). However, the distinction between different cognitive functions might be important to assess the patients' functional status appropriately and to determine treatment impact. To allow for such assessment, the aim of this study was to develop and psychometrically evaluate separate item banks for memory and attention based on the EORTC CAT Core CF item bank. METHODS: In a multistep process including an expert-based content analysis, we assigned 44 items from the EORTC CAT Core CF item bank to the memory or attention domain. Then, we conducted psychometric analyses based on a sample used within the development of the EORTC CAT Core CF item bank. The sample consisted of 1030 cancer patients from Denmark, France, Poland, and the United Kingdom. We evaluated measurement properties of the newly developed item banks using confirmatory factor analysis (CFA) and item response theory model calibration. RESULTS: Item assignment resulted in 31 memory and 13 attention items. Conducted CFAs suggested good fit to a 1-factor model for each domain and no violations of monotonicity or indications of differential item functioning. Evaluation of CATs for both memory and attention confirmed well-functioning item banks with increased power/reduced sample size requirements (for CATs ≥ 4 items and up to 40% reduction in sample size requirements in comparison to non-CAT format). CONCLUSION: Two well-functioning and psychometrically robust item banks for memory and attention were formed from the existing EORTC CAT Core CF item bank. These findings could support further research on self-reported cognitive functioning in cancer patients in clinical trials as well as for real-word-evidence. A more precise assessment of attention and memory deficits in cancer patients will strengthen the evidence on the effects of cancer treatment for different cancer entities, and therefore contribute to shared and informed clinical decision-making.

Achieving the UNAIDS 90-90-90 targets: a comparative analysis of four large community randomised trials delivering universal testing and treatment to reduce HIV transmission in sub-Saharan Africa.

BACKGROUND: Four large community-randomized trials examining universal testing and treatment (UTT) to reduce HIV transmission were conducted between 2012-2018 in Botswana, Kenya, Uganda, Zambia and South Africa. In 2014, the UNAIDS 90-90-90 targets were adopted as a useful metric to monitor coverage. We systematically review the approaches used by the trials to measure intervention delivery, and estimate coverage against the 90-90-90 targets. We aim to provide in-depth understanding of the background contexts and complexities that affect estimation of population-level coverage related to the 90-90-90 targets. METHODS: Estimates were based predominantly on "process" data obtained during delivery of the interventions which included a combination of home-based and community-based services. Cascade coverage data included routine electronic health records, self-reported data, survey data, and active ascertainment of HIV viral load measurements in the field. RESULTS: The estimated total adult populations of trial intervention communities included in this study ranged from 4,290 (TasP) to 142,250 (Zambian PopART Arm-B). The estimated total numbers of PLHIV ranged from 1,283 (TasP) to 20,541 (Zambian PopART Arm-B). By the end of intervention delivery, the first-90 target (knowledge of HIV status among all PLHIV) was met by all the trials (89.2%-94.0%). Three of the four trials also achieved the second- and third-90 targets, and viral suppression in BCPP and SEARCH exceeded the UNAIDS target of 73%, while viral suppression in the Zambian PopART Arm-A and B communities was within a small margin (~ 3%) of the target. CONCLUSIONS: All four UTT trials aimed to implement wide-scale testing and treatment for HIV prevention at population level and showed substantial increases in testing and treatment for HIV in the intervention communities. This study has not uncovered any one estimation approach which is superior, rather that several approaches are available and researchers or policy makers seeking to measure coverage should reflect on background contexts and complexities that affect estimation of population-level coverage in their specific settings. All four trials surpassed UNAIDS targets for universal testing in their intervention communities ahead of the 2020 milestone. All but one of the trials also achieved the 90-90 targets for treatment and viral suppression. UTT is a realistic option to achieve 95-95-95 by 2030 and fast-track the end of the HIV epidemic.

Developmental stage and morphology of the competent blastocyst are associated with sex of the child but not with other obstetric outcomes: a multicenter cohort study.

STUDY QUESTION: Are transfer day, developmental stage and morphology of the competent blastocyst in pregnancies leading to live birth associated with preterm birth, birthweight, length at birth and sex of the child? SUMMARY ANSWER: A high score in blastocyst developmental stage and in trophectoderm (TE) showed a significant association with the sex of the child, while no other associations with obstetric outcomes were observed. WHAT IS KNOWN ALREADY: The association between blastocyst assessment scores and obstetric outcomes have been reported in small single-center studies and the results are conflicting. STUDY DESIGN, SIZE, DURATION: Multicenter historical cohort study based on exposure data (transfer day (blastocyst developmental stage reached by Day 5 or Day 6)) blastocyst developmental stage (1-6) and morphology (TE and inner cell mass (ICM): A, B, C)) and outcome data (preterm birth, birthweight, length at birth, and sex of the child) from women undergoing single blastocyst transfer resulting in a singleton pregnancy and live birth. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from 16 private and university-based facilities for clinical services and research were used. A total of 7246 women, who in 2014-2018 underwent fresh-embryo transfer with a single blastocyst or frozen-thawed embryo transfer (FET) with a single blastocyst resulting in a singleton pregnancy were identified. Linking to the Danish Medical Birth Registry resulted in a total of 4842 women with a live birth being included. Cycles with pre-implantation genetic testing and donated gametes were excluded. The analyses were adjusted for female age (n = 4842), female BMI (n = 4302), female smoking (n = 4290), parity (n = 4365), infertility diagnosis (n = 4765), type of treatment (n = 4842) and center (n = 4842); some analyses additionally included gestational age (n = 4368) and sex of the child (n = 4833). MAIN RESULTS AND THE ROLE OF CHANCE: No statistically significant associations between blastocyst assessment scores (transfer day, developmental stage, TE, ICM) and preterm birth (8.3%) or birthweight (mean 3461.7 g) were found. The adjusted association between blastocysts with a TE score of C and a TE score of A and length at birth (mean 51.6 cm) were statistically significant (adjusted mean difference 0.4 cm (95% CI: 0.02; 0.77)). Blastocysts transferred with developmental stage score 5 compared to blastocysts transferred with score 3 had a 34% increased probability of being a boy (odds ratio (OR) 1.34 (95% CI: 1.09; 1.64). Further, TE score B blastocysts compared to TE score A blastocysts had a 31% reduced probability of being a boy (OR 0.69 (95% CI: 0.60; 0.80)). LIMITATIONS, REASONS FOR CAUTION: It is possible that some residual confounding remains. WIDER IMPLICATIONS OF THE FINDINGS: Blastocyst selection during ART does not appear to introduce any negative effects on obstetric outcome. Therefore, clinicians and patients can be reassured that the assessment scores of the selected blastocyst will not in themselves pose a risk of preterm birth or affect birthweight and the length at birth. STUDY FUNDING/COMPETING INTEREST(S): Unrestricted grant from Gedeon Richter Nordics AB, Sweden. None of the authors have any competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Inactivation of Cryptosporidium parvum oocysts and faecal indicator bacteria in cattle slurry by addition of ammonia.

AIMS: To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia. METHODS AND RESULTS: Escherichia coli, enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2 weeks in cattle slurry amended with 60 mmol l-1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P = 0·021) corresponding to 62·0% higher inactivation (P = 0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry. CONCLUSION: The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land. SIGNIFICANCE AND IMPACT OF THE STUDY: Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die-off rates of pathogens in organic waste can increase following increasing ammonia concentrations.

Expression-based analysis of genes related to single nucleotide polymorphism hits associated with bovine leukemia virus proviral load in Argentinean dairy cattle.

In dairy cattle infected with bovine leukemia virus (BLV), the proviral load (PVL) level is directly related to the viral transmission from infected animals to their healthy herdmates. Two contrasting phenotypic groups can be identified when assessing PVL in peripheral blood of infected cows. A large number of reports point to bovine genetic variants (single nucleotide polymorphisms) as one of the key determinants underlying PVL level. However, biological mechanisms driving BLV PVL profiles and infection progression in cattle have not yet been elucidated. In this study, we evaluated whether a set of candidate genes affecting BLV PVL level according to whole genome association studies are differentially expressed in peripheral blood mononuclear cells derived from phenotypically contrasting groups of BLV-infected cows. During a 10-mo-long sampling scheme, 129 Holstein cows were phenotyped measuring anti-BLV antibody levels, PVL quantification, and white blood cell subpopulation counts. Finally, the expression of 8 genes (BOLA-DRB3, PRRC2A, ABT1, TNF, BAG6, BOLA-A, LY6G5B, and IER3) located within the bovine major histocompatibility complex region harboring whole genome association SNP hits was evaluated in 2 phenotypic groups: high PVL (n = 7) and low PVL (n = 8). The log2 initial fluorescence value (N0) transformed mean expression values for the ABT1 transcription factor were statistically different in high- and low-PVL groups, showing a higher expression of the ABT1 gene in low-PVL cows. The PRRC2A and IER3 genes had a significant positive (correlation coefficient = 0.61) and negative (correlation coefficient = -0.45) correlation with the lymphocyte counts, respectively. Additionally, the relationships between gene expression values and lymphocyte counts were modeled using linear regressions. Lymphocyte levels in infected cows were better explained (coefficient of determination = 0.56) when fitted a multiple linear regression model using both PRRC2A and IER3 expression values as independent variables. The present study showed evidence of differential gene expression between contrasting BLV infection phenotypes. These genes have not been previously related to BLV pathobiology. This valuable information represents a step forward in understanding the BLV biology and the immune response of naturally infected cows under a commercial milk production system. Efforts to elucidate biological mechanisms leading to BLV infection progression in cows are valuable for BLV control programs. Further studies integrating genotypic data, global transcriptome analysis, and BLV progression phenotypes are needed to better understand the BLV-host interaction.

[Telemedicine in stroke-pertinent to stroke care in Germany]. / Telemedizin in der Schlaganfallversorgung ­ versorgungsrelevant für Deutschland.

BACKGROUND AND OBJECTIVE: Telemedical stroke networks improve stroke care and provide access to time-dependent acute stroke treatment in predominantly rural regions. The aim is a presentation of data on its utility and regional distribution. METHODS: The working group on telemedical stroke care of the German Stroke Society performed a survey study among all telestroke networks. RESULTS: Currently, 22 telemedical stroke networks including 43 centers (per network: median 1.5, interquartile range, IQR, 1-3) as well as 225 cooperating hospitals (per network: median 9, IQR 4-17) operate in Germany and contribute to acute stroke care delivery to 48 million people. In 2018, 38,211 teleconsultations (per network: median 1340, IQR 319-2758) were performed. The thrombolysis rate was 14.1% (95% confidence interval 13.6-14.7%) and transfer for thrombectomy was initiated in 7.9% (95% confidence interval 7.5-8.4%) of ischemic stroke patients. Financial reimbursement differs regionally with compensation for telemedical stroke care in only three federal states. CONCLUSION: Telemedical stroke care is utilized in about 1 out of 10 stroke patients in Germany. Telemedical stroke networks achieve similar rates of thrombolysis and transfer for thrombectomy compared with neurological stroke units and contribute to stroke care in rural regions. Standardization of network structures, financial assurance and uniform quality measurements may further strengthen the importance of telestroke networks in the future.

In vivo fluorescence molecular tomography of induced haemarthrosis in haemophilic mice: link between bleeding characteristics and development of bone pathology.

BACKGROUND: Haemophilic arthropathy is a chronic and debilitating joint disease caused by recurrent spontaneous joint bleeds in patients with haemophilia. Understanding how characteristics of individual joint bleeds relate to the subsequent development of arthropathy could improve management and prevention of this joint disease. Here, we aimed to explore relations between joint bleed characteristics and development of bone pathology in a mouse model of haemophilic arthropathy by using novel in vivo imaging methodology. METHODS: We characterised induced knee bleeds in a murine model of haemophilic arthropathy by quantitative in vivo fluorescence molecular tomography (FMT) and by measurements of changes in the diameter of the injured knee. Wild-type mice and non-injured haemophilic mice acted as controls. Development of arthropathy was characterised by post mortem evaluation of bone pathology by micro-CT 14 days after bleed-induction. In an in vitro study, we assessed the effect of blood on the quantification of fluorescent signal with FMT. RESULTS: In most injured haemophilic mice, we observed significant loss of trabecular bone, and half of the mice developed pathological bone remodelling. Development of pathological bone remodelling was associated with significantly increased fluorescent signal and diameter of the injured knee just 1 day after induction of the bleed. Further, a correlation between the fluorescent signal 1 day after induction of the bleed and loss of trabecular bone reached borderline significance. In the in vitro study, we found that high concentrations of blood significantly decreased the fluorescent signal. CONCLUSION: Our results add novel insights on the pathogenesis of haemophilic arthropathy and underline the importance of the acute phase of joint bleeds for the subsequent development of arthropathy.

New insights into early recovery after robotic surgery for endometrial cancer.

OBJECTIVE: To assess early recovery of physical health after robotic minimally invasive surgery (RMIS) for early-stage endometrial cancer using the European Organisation of Research and Treatment of Cancer Computer Adaptive Test Core questionnaire (EORTC CAT Core). The EORTC CAT Core provides individualised measurements while maintaining comparability. A hypothesis of individual complete recovery to baseline within three post-surgical weeks was evaluated. METHODS: Ninety-four women who underwent RMIS for early-stage endometrial cancer were included consecutively. The EORTC CAT Core was distributed before surgery and prospectively every week during the first post-operative month. Repeated measures models were fitted for each of the four domains (physical functioning, role function, fatigue, and pain) and tested for impact of age, ASA score, minor/major surgery, and the individual baseline scores (poorest, intermediate, best). RESULTS: Women with the lowest physical functioning, lowest role function, highest fatigue level, and highest pain level at baseline all recovered within three weeks. Women with the highest physical functioning, highest role function, lowest level of fatigue, and lowest level of pain at baseline did not reach their individual baselines within the first post-operative month but had the most favourable domain-scores three weeks post-operatively. CONCLUSION: The individual woman's physical health baseline score is predictive for her postoperative recovery following RMIS for early-stage endometrial cancer. Women with the best physical health had the best postoperative functions and lowest level of symptoms; however their recovery to baseline was prolonged. Computer adaptive testing may be a valuable tool for individualised pre-operative information and supportive care during surveillance.

The healthy donor effect impacts self-reported physical and mental health - results from the Danish Blood Donor Study (DBDS).

AIMS: This study aimed at quantifying the healthy donor effect by comparing self-perceived mental and physical health between blood donors and non-donors. BACKGROUND: In theory, the selection process known as the healthy donor effect should result in better self-perceived, health-related quality of life in donors than in non-donors. METHODS: The Short Form-12 data from the Danish Twin Registry (DTR) was compared with the data from the Danish Blood Donor Study (DBDS). Data on age, sex and smoking status were included in the analyses. The multivariable linear regression analysis was stratified by sex and age group intervals. Outcome variables were the mental component score (MCS) and the physical component score (PCS). RESULTS: A total of 28 982 and 36 913 participants from the DTR and the DBDS, respectively, were included in this study. Younger donors had higher MCS than non-donors, whereas MCS was only marginally high in older donors compared with non-donors. In contrast, PCS was almost similar for both young donors and non-donors. With the increase in age, non-donors had lower PCS than donors. CONCLUSIONS: Two selection patterns were revealed. Among young individuals, better self-perceived mental health was associated with a blood donor. With the increase in age, better self-perceived physical health was associated with blood donation.

Fundus autofluorescence and spectral domain optical coherence tomography as predictors for long-term functional outcome in rhegmatogenous retinal detachment.

PURPOSE: To detect pre- and postoperative retinal changes in fundus autofluorescence (AF) and spectral domain optical coherence tomography (SD-OCT) and to correlate these with functional outcome in patients with primary rhegmatogenous retinal detachment (RRD). METHODS: A prospective, 30-month study of patients operated with 25-gauge vitrectomy for primary RRD. Patients were examined preoperatively and after 6 and 30 months, using ultrawide-field AF images (UWFI) (Optos 200Tx) and SD-OCT (Topcon 3D OCT-2000) imaging. RESULTS: Of 84 patients (84 eyes) included at baseline, 100.0 and 86.9% were re-examined at month 6 and 30, respectively. Preoperative findings such as macular attachment, detachment > 750 µm from foveola, lack of intraretinal separation, and subfoveal elevation ≤ 500 µm were all associated with better BCVA at months 6 and 30. Postoperative disruption of the photoreceptor layer was associated with poor BCVA at month 6 (p < 0.001) but not at month 30. At baseline, AF-demarcation of RRD was demonstrated by a hyperfluorescent edge in 92.0% and was associated with visual impairment at months 6 (p = 0.003) and 30 (p = 0.003). Visual outcome at month 30 was good (≤ 0.3 logMAR (≥ 20/40 Snellen)), regardless of the preoperative, macular status. However, with significantly better visual outcome in patients with macula attachments versus partly or totally macular detachments (p < 0.001). CONCLUSION: Fundus AF and SD-OCT is able to identify retinal reestablishment up to 30 months after primary RRD, with good correlation to BCVA. These findings emphasize the importance of long-term studies for final visual recovery.

Effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity: a randomized controlled trial.

BACKGROUND: Aerobic exercise is recommended for weight management but energy balance is often less negative than predicted from exercise energy expenditure (ExEE). OBJECTIVE: To examine effects of active commuting and leisure-time exercise on fat loss in women and men with overweight and obesity. METHODS: We randomized 130 younger, physically inactive women and men with overweight and obesity (body mass index: 25-35 kg m-2) to 6 months of habitual lifestyle (control; CON, n=18), active commuting (BIKE, n=35) or leisure-time exercise of moderate (MOD, 50% VO2peak reserve, n=39) or vigorous intensity (VIG, 70% VO2peak reserve, n=38). The primary outcome was change in fat mass measured by dual-energy X-ray absorptiometry, which was analyzed intention-to-treat. Accumulated energy balance was calculated based on changes in body composition, and ExEE was calculated based on heart rate monitoring during exercise. RESULTS: Testing at 3 and 6 months was completed by 95 and 90 participants, respectively. Fat mass was reduced after 3 and 6 months in BIKE (3 months: -3.6 (-5.5; -1.7) kg (mean (95% CI)); 6 months: -4.2 (-6.6; -1.9) kg; both: P<0.001), MOD (3 months: -2.2 (-3.9; -0.4) kg; 6 months: -2.6 (-4.8; -0.5) kg, both: P<0.02) and VIG (3 months: -3.4 (-5.2; -1.7) kg; 6 months: -4.5 (-6.6; -2.3) kg; both: P<0.001) compared with CON. Furthermore, fat loss was greater in VIG compared with MOD (6 months: -1.8 (-3.6; -0.1) kg, P=0.043). Based on the ExEE and the accumulated energy balance MOD compensated for the ExEE (77 (48; 106) %) but not BIKE (38 (-18; 95) %) and VIG (21 (-14; 55) %). CONCLUSIONS: A meaningful fat loss was obtained by 6 months of active commuting and leisure-time exercise, but fat loss was greater with vigorous compared with moderate intensity exercise. Active commuting is an alternative to leisure-time exercise in the management of overweight and obesity. The trial was registered at clinicaltrials.gov as NCT01962259 (main trial) and NCT01973686 (energy metabolism sub-study).

Targeted gene sequencing and whole-exome sequencing in autopsied fetuses with prenatally diagnosed kidney anomalies.

Identification of fetal kidney anomalies invites questions about underlying causes and recurrence risk in future pregnancies. We therefore investigated the diagnostic yield of next-generation sequencing in fetuses with bilateral kidney anomalies and the correlation between disrupted genes and fetal phenotypes. Fetuses with bilateral kidney anomalies were screened using an in-house-designed kidney-gene panel. In families where candidate variants were not identified, whole-exome sequencing was performed. Genes uncovered by this analysis were added to our kidney panel. We identified likely deleterious variants in 11 of 56 (20%) families. The kidney-gene analysis revealed likely deleterious variants in known kidney developmental genes in 6 fetuses and TMEM67 variants in 2 unrelated fetuses. Kidney histology was similar in the latter 2 fetuses-presenting a distinct prenatal form of nephronophthisis. Exome sequencing identified ROBO1 variants in one family and a GREB1L variant in another family. GREB1L and ROBO1 were added to our kidney-gene panel and additional variants were identified. Next-generation sequencing substantially contributes to identifying causes of fetal kidney anomalies. Genetic causes may be supported by histological examination of the kidneys. This is the first time that SLIT-ROBO signaling is implicated in human bilateral kidney agenesis.

Epidemiology of cutaneous lupus erythematosus and the associated risk of systemic lupus erythematosus: a nationwide cohort study in Denmark.

Objectives The objectives of this paper are to describe the epidemiology of cutaneous lupus erythematosus (CLE) and its subtypes in Denmark, and to investigate the probability of receiving a subsequent diagnosis of systemic lupus erythematosus (SLE) and the related time course. Methods A nationwide registry-based cohort study was conducted in Denmark based on data from the Danish National Patient Registry from 1998 to 2013 using International Classification of Diseases, Revision 10. Results We identified 2380 patients with CLE. The annual incidence rate (IR) of CLE was 2.74/100,000 with a female:male ratio of 4:1. During 12,047 person-years of follow-up, 8% were diagnosed with SLE. The probability of receiving a subsequent diagnosis of SLE was 12.9% after 10 years taking death as a competing risk into consideration, and the probability was highest among women and patients diagnosed with subacute CLE. The median time until a diagnosis of SLE was 2.05 years. Conclusions This is the first nationwide study on CLE in Denmark. Although we found the annual IR of CLE and the risk of receiving an additional diagnosis of SLE to be lower than previously described, continued monitoring and thorough information for patients with CLE is important due to the inherent risk of SLE.

Untargeted antifungal therapy in adult patients with complicated intra-abdominal infection: a systematic review.

BACKGROUND: Systematic reviews comparing untargeted antifungal treatment with placebo or no treatment in critically ill patients have provided conflicting results. We aimed to assess patient-important benefits and harms of untargeted antifungal therapy vs. placebo or no treatment in adult patients with complicated intra-abdominal infection. METHODS: We conducted a systematic review with meta-analysis and trial sequential analysis of randomised clinical trials assessing untargeted antifungal therapy compared to placebo or no treatment in adults with complicated intra-abdominal infection. We used the Cochrane and GRADE methodologies and exclusively assessed patient-important outcomes. Two independent authors screened trials for eligibility, extracted data and assessed risk of bias. We performed conventional meta-analyses, including sensitivity and subgroup analyses, and trial sequential analysis to assess the risk of random errors and to estimate trial sequential analysis adjusted confidence intervals. RESULTS: We included six trials (1,067 patients) in the review, and four trials reported data on the predefined outcome measures and were included in the meta-analysis. Three of the four trials had high risk of bias. We observed no statistically significant difference in mortality (relative risk 0.58, 95% confidence interval 0.24-1.39) or in any of the other patient-important outcomes between untargeted antifungal treatment and placebo or no treatment (low/very low quality of evidence). Trial sequential analysis demonstrated lack of data and high risk of random errors. CONCLUSIONS: The quantity and quality of evidence supporting untargeted antifungal treatment in adult patients with complicated intra-abdominal infection are low to very low with no firm evidence for benefit or harm.

Empirical metronidazole for patients with severe bacterial infection: protocol for a systematic review.

INTRODUCTION: Anaerobic bacteria are believed to be common pathogens in severe infections. Yet, they are difficult to culture and consequently often unrecognised in clinical infections. Metronidazole is often used empirically for potential anaerobic infections, as the resistance to metronidazole is low. However, disadvantages of metronidazole use exist, including drug interactions, side effects and economical expenses. Currently, the balance between the benefits and harms of empirical metronidazole for severe bacterial infections is unknown. We aim to assess patient-important benefits and harms of empirical metronidazole vs. placebo or no treatment in adult patients with severe bacterial infection of any origin in a systematic review of randomised clinical trials with meta-analysis and trial sequential analysis. METHODS AND ANALYSIS: This protocol provides details on the planned systematic review, which will be prepared according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement, the Cochrane Handbook, and the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) criteria. The primary outcome is all-cause mortality. Secondary outcomes include adverse events, secondary infections, use of life support, antibiotic resistance and hospital length of stay. We will conduct conventional meta-analyses, including predefined subgroup- and sensitivity analyses. Additionally, we will assess the risk of random errors by trial sequential analysis. ETHICS AND DISSEMINATION: Ethical approval is not needed, as the outlined review exclusively will include previously published data. We aim to publish in an international, peer-reviewed journal.

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs.