Day 1 Research Articles

In severe alcohol-related hepatitis, acute kidney injury is prevalent, associated with mortality independent of liver disease severity, and can be predicted using IL-8 and micro-RNAs.

The prevalence, prediction and impact of acute kidney injury (AKI) in alcohol-related hepatitis (AH) is uncertain. We aimed to determine AKI incidence; association with mortality; evaluate serum biomarkers and the modifying effects of prednisolone and pentoxifylline in the largest AH cohort to date. Participants in the Steroids or Pentoxifylline for Alcoholic Hepatitis trial with day zero (D0) creatinine available were included. AKI was defined by modified International Club of Ascites criteria; incident AKI as day 7 (D7) AKI without D0-AKI. Survival was compared by Kaplan-Meier; mortality associations by Cox regression; associations with AKI by binary logistic regression; biomarkers by AUROC analyses. D0-AKI was present in 198/1051 (19%) participants; incident AKI developed in a further 119/571 (21%) with available data. Participants with D0-AKI had higher 90-day mortality than those without (32% vs. 25%, p = 0.008), as did participants with incident AKI compared to those without D0-AKI or incident AKI (47% vs. 25%, p < 0.001). Incident AKI was associated with D90 mortality adjusted for age and discriminant function (AHR 2.15, 1.56-2.97, p < 0.001); D0-AKI was not. Prednisolone therapy reduced incident AKI (AOR 0.55, 0.36-0.85, p = 0.007) but not mortality. D0 bilirubin and IL-8 combined, miR-6826-5p, and miR-6811-3p predicted incident AKI (AUROCs 0.726, 0.821, 0.770, p < 0.01). Incident AKI is associated with 90-day mortality independent of liver function. Prednisolone therapy was associated with reduced incident AKI. IL-8 and several miRNAs are potential biomarkers to predict AKI. Novel therapies to prevent incident AKI should be evaluated in AH to reduce mortality.

Is Carboxyhaemoglobin an Effective Bedside Prognostic Tool for Sepsis and Septic Shock Patients?

Proper management of sepsis poses a challenge even today, with early diagnosis and targeted treatment being the most important steps. Easy, cost-effective bedside tools are needed in order to pinpoint towards the outcome of sepsis or septic shock. This study aims to find a correlation between Sequential Organ Failure Assessment (SOFA), Acute Physiology and Chronic Health Evaluation II (APACHE II) and Simplified Acute Physiology Score II (SAPS II) severity scores, the Neutrophil-Lymphocytes Ratio (NLR) and carboxyhaemoglobin (COHb) levels in septic or septic shock patients with the scope of establishing a bed side cost-effective prognostic tool. A pilot, prospective, observational, and ongoing study was conducted on 61 patients admitted with sepsis or septic shock according to the SEPSIS 3 Consensus definition. We followed clinical and paraclinical parameters on day 1 (D1) and day 5 (D5) after meeting the inclusion criteria. On D1 we found a statistically significant positive correlation between each severity score (p <0.0001), r = 0.7287 for SOFA vs. APACHE II with CI: 0.5841-0.8285, r = 0.6862 for SOFA vs. SAPS II with CI: 0.5251-0.7998 and r = 0.8534 for APACHE II vs. SAPS II with CI: 0.7663 to 0.9097. On D5 we observed similar results: a significant positive correlation between each severity score (p <0.0001), with r = 0.7877 for SOFA vs. APACHE II with CI: 0.6283 to 0.8836, r = 0.8210 for SOFA vs. SAPS II with CI: 0.6822 to 0.9027 and r = 0.8880 for APACHE II vs. SAPS II., CI: 0.7952 to 0.9401. Nil correlation was found between the severity scores, NLR and COHb on D1 and D5. Cost-effective bedside tools to pinpoint towards the outcome of sepsis are yet to be found, however the positive correlation between the severity scores point out to a combination of such tools for prognosis prediction of septic or septic shock patients.

Twenty-Four Percent of Wildland Firefighters Reach Critical Levels of Serum Creatine Kinase During 80-Hour Critical Training.

Wildland firefighters (WLFFs) must undergo a 2-wk critical training (CT) period prior to deployment to the field. This stress may result in clinical risks, including severe muscle damage and rhabdomyolysis. We aimed to document the effects of WLFFs' CT on physiologic markers of muscle damage and soreness. Two interagency hotshot crews (n=25) were followed during spring 2022 for 80 h of training. Activity counts as well as records of upper-body (US) and lower-body (LS) muscle soreness were collected daily. Body weight (BW) and skinfold measurements were recorded on Day 1 (D1) and D11 to estimate body fat (BF) and lean body weight (LBW). Blood was collected on D1 and D11 to measure muscle and liver damage markers. Critical training resulted in significant elevations in creatine kinase (CK) (216.9±57.4 U/L vs 5166.4±1927.8 U/L, P=0.017) and lactate dehydrogenase (LDH) (175.5±4.0 IU/L vs 340.0±42.6 IU/L, P=0.001) despite no significant changes in BW, BF, LBW, cortisol, or testosterone. Main effects of time were seen in US and LS, peaking on D11 (US: 5.2±0.4 cm, P<0.001; LS: 5.5±0.4 cm, P<0.001). Those who spent the most minutes with activity counts of >1500 counts/min showed the greatest increase in CK and LDH. These data suggest that WLFFs undergo significant physiologic stressors, resulting in muscle soreness and damage during CT, with 6 of the 25 subjects reaching critical levels of serum CK. It appears that much of the muscle damage and soreness occurred because of unaccustomed WLFF job-specific tasks.

Comprehensive transcriptional analysis of pig facial skin development.

Skin development is a complex process that is influenced by many factors. Pig skin is used as an ideal material for xenografts because it is more anatomically and physiologically similar to human skin. It has been shown that the skin development of different pig breeds is different, and some Chinese pig breeds have the characteristics of skin thickness and facial skin folds, but the specific regulatory mechanism of this skin development is not yet clear. In this study, the facial skin of Chenghua sows in the four developmental stages of postnatal Day 3 (D3) , Day 90 (D90) , Day 180 (D180), and Year 3 (Y3) were used as experimental materials, and RNA sequencing (RNA-seq) analysis was used to explore the changes in RNA expression in skin development at the four developmental stages, determine the differentially expressed messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), and perform functional analysis of related genes by Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. A pairwise comparison of the four developmental stages identified several differentially expressed genes (DEGs) and found that the number of differentially expressed RNAs (DE RNAs) increased with increasing developmental time intervals. Elastin (ELN) is an important component of the skin. Its content affects the relaxation of the epidermis and dermal connection, and its expression is continuously downregulated during the four developmental stages. The functions of DEGs at different developmental stages were examined by performing GO and KEGG analyses, and the GO terms and enrichment pathways of mRNAs, lncRNAs, miRNAs, and circRNAs highly overlapped, among which the PPAR signaling pathway, a classical pathway for skin development, was enriched by DEGs of D3 vs. D180, D90 vs. D180 and D180 vs. Y3. In addition, we constructed lncRNA-miRNA-mRNA and circRNA-miRNA interaction networks and found genes that may be associated with skin development, but their interactions need further study. We identified a number of genes associated with skin development, performed functional analyses on some important DEGs and constructed interaction networks that facilitate further studies of skin development.

Prevalence, Risk factors, and Shedding of Human Herpes Virus-8 among Men Having Sex With Men Enrolled in a Pre-exposure Prophylaxis Study.

Seroprevalence and risk factors for Human Herpesvirus-8 (HHV-8) infection among HIV-negative men who have sex with men (MSM) on pre-exposure prophylaxis (PrEP) have not been well characterized. Our objectives were to assess the prevalence and incidence of HHV-8 infection in MSM enrolled on PrEP and assess viral shedding in seropositive participants. The ANRS IPERGAY study enrolled 429 participants in France and Canada to evaluate oral PrEP for HIV-1 prevention. Stored sera samples at day 0 (D0) and last visit were tested for the detection of HHV-8 antibodies using an indirect immunofluorescence assay. Baseline characteristics were analyzed to identify risk factors associated with HHV-8 seropositivity. Among seropositive participants, HHV-8 DNA was quantified on available oral and anal swabs, and ORF-K1 typing performed on HHV-8 positive samples. One hundred participants were seropositive at D0 (prevalence of 24%, 95% Confidence Interval (95%CI): 20·0-28·4) and 18/329 seroconverted during the study (incidence rate of 2·66 per 100 person-years, 95%CI: 1·57-4·20). Risk factors independently associated with baseline HHV-8 seropositivity included older age, high number of sexual partners, chemsex use and HSV-2 seropositivity. Among HHV-8 seropositive participants with available swab(s) for virological analysis, 37/115 (32%) displayed HHV-8 oral shedding, and 5/113 (4.4%) anal shedding at least once. Four patients had positive viral load before seroconversion. Prevalence and incidence of HHV-8 infection were high in HIV-negative PrEP users. Among seropositive participants, HHV-8 DNA is mainly detected in saliva, which may play a major role in viral transmission in this population.

Morphological Evaluation of Transscleral Laser Retinopexy in Rabbits: Comparison of Optical Coherence Tomography and Histologic Examinations.

Transscleral retinopexy is a preventive technique used against retinal detachment. Fundus examination can allow the monitoring of morphological retinal changes in the progression of photocoagulation lesions, without offering details on the morphological changes by the retinal lesion. The aim of the study was to assess the progression of photocoagulation lesions induced by transscleral retinopexy (840 nm diode laser), by comparing the optical coherence tomography (OCT) and histological images over a period of six weeks on eight pigmented New Zealand healthy rabbits (four males and four females; n = 16 eyes). All rabbits underwent transscleral retinopexy on their left eye on day 0 (D0). Measurements of the photocoagulation lesions were obtained in vivo on D0, D7, D15, D21, and D42 by acquiring OCT images of both eyes from all rabbits. On D1, D7, D21, and D42, two rabbits were euthanized, and their eyes were enucleated. A significant effect by time on the decrease in the central retinal thickness of the photocoagulation lesion was observed from D1 to D7 (p = 0.001); however, no such effect was observed on the horizontal length ((HL) p = 0.584) of the lesion surface. The reliability between the OCT and histological measurements, which were evaluated using intraclass correlation coefficients, was excellent for measuring the retinal thickness at the center (ICC = 0.91, p < 0.001), moderate for the right side of the retinal lesions (ICC = 0.72, p = 0.006), and not significant for the left side and HL (p = 0.055 and 0.500, respectively). The morphological changes observed in the OCT and histopathological images of the photocoagulation lesions were qualitatively described over time. OCT is an effective tool for monitoring changes in photocoagulation lesions. Some measurements and qualitative changes showed an adequate correlation between the OCT and histological findings.

Plasma estradiol profile after administration of different types of estradiol esters in acyclic mares.

Although the use of different estradiol esters has been extensively studied in hormonal protocols in cows, such information is lacking in mares. The present study aimed to assess the effects of treatment with the same doses and administration frequency of estradiol cypionate, estradiol benzoate and 17β estradiol on plasma estradiol (E2) concentrations of acyclic mares and correlate the E2 profile to the endometrial edema score. Sixteen treatments were performed in 14 mares randomly divided into three groups: EB (n=5), EC (n=5) and 17β (n=6), receiving 10mg on day 0 (D0), 6mg on D1 and 4mg on D2 of estradiol benzoate, estradiol cypionate, and estradiol 17β, respectively. Blood samples, rectal palpations and ultrasound evaluations were performed once daily, starting before the first estradiol treatment (D0) until edema disappearance or D8. Moderate to high edema was observed in all groups 24h after the first estradiol administration. Edema persisted above score 2 until D7, D5 and D4 in groups EC, EB and 17β, respectively. Higher edema was found on D2 in EB group compared to 17β, on D6 and D7 in EC compared to 17β, and on D8 in EC compared to EB (p≤0.05). Maximum E2 concentrations were detected on D1 in groups: EB, showing a sharp decrease from D2 to D3 (p<0.0001); and 17β, where no differences were observed between treatment days (p≥0.05). In the EC group, maximum concentrations were observed on D2, which remained high from D2 to D4 compared to the other days (p<0.0001). Plasma concentration of E2 was higher in EB than the other groups on D0 and D1, and EB>EC>17β on D2. Plasma E2 concentrations and edema score were positively correlated, being moderate in groups EB and EC and weak in the 17β group. In conclusion, the most pronounced plasma E2 concentration was observed in EB group 24h after the administration of the first dose. Estradiol concentrations peaked 48h after EC administration, while a distinct peak was not observed in a 24h interval evaluation in the 17β group. Moreover, high edema does not necessarily reflect on high E2 concentrations in acyclic mares.

Temporada corta de reprodución controlada com ovejas Santa Inês criadas em clima tropical húmedo

The objective of this work was to evaluate the adoption of management techniques that could allow for a shorter controlled breeding season. In three consecutive mating seasons (MS1: September/2021, n=45; MS2: June/2022, n=42 and MS3: March/2023, n=43), lactating Santa Ines ewes, multiparous, aged between two and four years, mean live weight of 62.20 ± 4.60 kg and mean body condition score of 3.4 ± 0.4 (scale 1-5), two vasectomized males and two breeding rams with prior andrological evaluation, one Santa Ines and one Dorper. A pre-mating period was implemented with biostimulation using vasectomized males for 21 days. After this period, on day 0 (D0), an IM injection of 120.5µg of cloprostenol, a prostaglandin F2alpha analogue (PGF2α), was performed and estrus was observed until D3; ewes with identified estrus were mated by the breeding rams, as per controlled breeding. Ewes that did not show estrus were submitted to a second IM injection of PGF2α seven days after the first dose (D7), then repeating the management of estrus observation and controlled mating. Ewes were allowed three attempts at conception. Pregnancy diagnosis was performed by ultrasonography (D30). During biostimulation, 100% of the ewes showed estrus in the three mating seasons. There was no significant difference in performance between seasons (P&gt;0.05). From the total of ewes in season (n= 130), 90.0% (n=117) became pregnant, 71.5% (n=93) in the first, 13.9% (n=18) in the second and 4.6% (n= 6) in the third mating. Prior biostimulation was efficient in inducing estrus to lactating Santa Ines ewes and the use of PGF2α was sufficient to synchronize estrus and generate a shorter controlled breeding season with up to three matings, presenting an efficient method to concentrate births and standardize allotments of lambs in a production system under humid tropical climate.

Chloroquine inhibiting the activation of primary hepatic stellate cells induced by lipopolysaccharide

Lipopolysaccharide (LPS) leakage from bacteria causes liver inflammation and directly activates hepatic stellate cells (HSCs), contributing to liver fibrosis development. The current study investigated the effect of an autophagy inhibitor (Chloroquine, CQ) on in vitro HSC activation induced by LPS. Primary HSCs were isolated and cultured from BALB/c mice. Cells were cultured for 24 hours in a medium containing LPS 200 ng/ml, CQ 5 μM, or LPS 200 ng/ml + CQ 5 μM. All treated cells were evaluated by immunocytochemistry for morphology, proliferation, expression of activation markers α-sma and collagen 1, storage of lipid droplets and autophagy markers lc3/p62 at days 1 (D1), 3 (D3) and 7 (D7) after treatment. Proliferation of primary HSCs and expression of activation markers (α-sma and collagen 1) caused by LPS induction were significantly inhibited by CQ treatment until D7 of culture (p&lt;0.01). Additionally, CQ treatment preserves cell function in lipid droplet storage. This effect of CQ on LPS-induced HSC activation was consistent with the late-stage inhibition of autophagy demonstrated by the decrease in lc3 and increase in p62 at D1 (p&lt;0.01). CQ inhibits primary HSC activation in vitro when induced by LPS.

Prognostic and predictive value of endothelial dysfunction biomarkers in sepsis-associated acute kidney injury: risk-stratified analysis from a prospective observational cohort of pediatric septic shock

BackgroundSepsis-associated acute kidney injury (SA-AKI) is associated with high morbidity, with no current therapies available beyond continuous renal replacement therapy (CRRT). Systemic inflammation and endothelial dysfunction are key drivers of SA-AKI. We sought to measure differences between endothelial dysfunction markers among children with and without SA-AKI, test whether this association varied across inflammatory biomarker-based risk strata, and develop prediction models to identify those at highest risk of SA-AKI.MethodsSecondary analyses of prospective observational cohort of pediatric septic shock. Primary outcome of interest was the presence of ≥ Stage II KDIGO SA-AKI on day 3 based on serum creatinine (D3 SA-AKI SCr). Biomarkers including those prospectively validated to predict pediatric sepsis mortality (PERSEVERE-II) were measured in Day 1 (D1) serum. Multivariable regression was used to test the independent association between endothelial markers and D3 SA-AKI SCr. We conducted risk-stratified analyses and developed prediction models using Classification and Regression Tree (CART), to estimate risk of D3 SA-AKI among prespecified subgroups based on PERSEVERE-II risk.ResultsA total of 414 patients were included in the derivation cohort. Patients with D3 SA-AKI SCr had worse clinical outcomes including 28-day mortality and need for CRRT. Serum soluble thrombomodulin (sTM), Angiopoietin-2 (Angpt-2), and Tie-2 were independently associated with D3 SA-AKI SCr. Further, Tie-2 and Angpt-2/Tie-2 ratios were influenced by the interaction between D3 SA-AKI SCr and risk strata. Logistic regression demonstrated models predictive of D3 SA-AKI risk performed optimally among patients with high- or intermediate-PERSEVERE-II risk strata. A 6 terminal node CART model restricted to this subgroup of patients had an area under the receiver operating characteristic curve (AUROC) 0.90 and 0.77 upon tenfold cross-validation in the derivation cohort to distinguish those with and without D3 SA-AKI SCr and high specificity. The newly derived model performed modestly in a unique set of patients (n = 224), 84 of whom were deemed high- or intermediate-PERSEVERE-II risk, to distinguish those patients with high versus low risk of D3 SA-AKI SCr.ConclusionsEndothelial dysfunction biomarkers are independently associated with risk of severe SA-AKI. Pending validation, incorporation of endothelial biomarkers may facilitate prognostic and predictive enrichment for selection of therapeutics in future clinical trials among critically ill children.Graphical abstract

Adjuvant Lipoic acid Injection in Sepsis treatment in China (ALIS study): protocol for a randomised, single-blind, placebo-controlled trial

IntroductionSepsis is a life-threatening immune disorder resulting from an dysregulated host response to infection. Adjuvant therapy is a valuable complement to sepsis treatment. Lipoic acid has shown potential in attenuating...

CD12 Citalopram-induced drug reaction with eosinophilia and systemic reaction confirmed by patch testing

Abstract A 46-year-old man was referred for investigation of an acute widespread rash and fever that started 4 weeks after commencing citalopram. Clinical and histological features were consistent with drug reaction with eosinophilia and systemic reaction (DRESS). He was not on any other medication, had no known allergies and had not previously taken a selective serotonin reuptake inhibitor (SSRI). Six months after resolution, he was patch tested to the extended British Baseline Series and citalopram (crushed tablet in 50% petrolatum). Readings on days 2 (D2) and 4 (D4) were positive for citalopram (D2 ++ and D4 +++). To investigate possible crossreactivity, he was subsequently patch tested to fluoxetine, paroxetine and sertraline (crushed tablets in 50% petrolatum), which showed a positive reaction to paroxetine [D2 (–) and D4 (+)]. Patch tests to citalopram and paroxetine were negative with no irritancy in a normal volunteer. SSRIs are extensively and increasingly used for depression and other mental health problems; 45 000 000 UK prescriptions were issued in the last financial year. Despite such widespread use, cutaneous adverse drug reactions (CADRs) appear to be rare. From 1998 to 2022, the Medicines and Healthcare products Regulatory Agency recorded 16 586 adverse reactions to citalopram, of which only 1077 (6.5%) were cutaneous. Various rashes have been reported to arise from SSRI treatment, ranging from maculopapular eruptions to toxic epidermolysis necrolysis/Stevens–Johnson syndrome. There have been isolated reports of DRESS from fluoxetine, sertraline and escitalopram (the S-enantiomer of citalopram). To our knowledge, this is the first reported case of citalopram-induced DRESS. The sensitivity and specificity of patch tests in the diagnosis of delayed CADR varies according to the drug and rash. We have found case reports of positive patch tests to sertraline and paroxetine following maculopapular exanthems. Our findings highlight a diagnostic role for patch tests with an extemporaneous preparation in SSRI-induced DRESS. SSRIs have diverse chemical structures, and we speculate that patch tests may be of benefit in identifying a safer alternative should further SSRI treatment be needed.

Comparison of the local safety of two multi-component feline vaccines, adjuvanted (1 mL) versus non-adjuvanted at reduced volume (0.5 mL), using computed tomography imaging

In 2020, a new 0.5 mL presentation of PUREVAX® RCP FeLV was registered and introduced in Europe.The objectives of this study were to investigate the local safety of this non-adjuvanted vaccine at reduced volume by classical methods (clinical examination, histopathology) and to evaluate the suitability of an alternative non-invasive methodology, the computed tomography (CT). For this purpose, the course of local reactions was assessed for 3 months after subcutaneous injection of PUREVAX® RCP FeLV 0.5 mL and compared to an adjuvanted vaccine, LEUCOFELIGEN® FeLV/RCP 1.0 mL.Injection site reactions consisted mainly of swelling reactions, which were more frequent, more pronounced and long-lasting in the adjuvanted vaccine group. Microscopically, in this group, moderate to severe inflammatory reactions were observed on day 7 (D7) and D21 post-injection and still present on D84, while mild inflammatory lesions were observed in the non-adjuvanted vaccine group only on D7 and D21. With the adjuvanted vaccine, inflamed areas were measurable by CT scan in all cats on D7 and D21, whereas they were detected only on D7 and only in 20 % of cats from the non-adjuvanted vaccine group. Besides the higher frequency, the mean inflamed volume was nearly 300 times larger in adjuvanted vaccine group on D7.Using different methodologies, the favorable safety profile of PUREVAX® RCP FeLV 0.5 mL was confirmed. Furthermore, the vaccine is aligned with current vaccination guidelines by inducing less inflammatory reactions, being adjuvant-free and injectable under a reduced volume, thus improving the convenience of administration in recommended sites (eg, legs).CT scan proved to be a suitable non-invasive method for the experimental follow-up of injection site reactions, yielding results consistent with clinical assessment and histopathology on D7 and D21. CT scan substantiated large differences between the investigated vaccines with a more prominent inflammatory reaction after injection of an adjuvanted vaccine.

O-162 Fresh or frozen Day 6 blastocyst transfer : Is there still a question?

Abstract Study question Whether slow developing expanded day 6 (D6) blastocysts should be either transferred during fresh cycle or systematically vitrified at D6 Summary answer Fresh D6 blastocyst transfer is independently associated with reduced live birth rate compared to frozen thawed D6 transfer What is known already Live birth rate (LBR) after D5 blastocyst transfers are significantly higher when compared with D6 embryos in both fresh and frozen-vitrified embryo transfer cycles according to the last published meta-analyses. Therefore, for women obtaining only D6 blastocysts, chances of pregnancy may be lower but still persist and these embryos should be transferred. The question of the best strategy for transfer (i.e. in fresh or frozen cycles) remains unclear while data on this subject are scarce. Study design, size, duration Retrospective observational cohort study. Patients having only embryos reaching blastocyst stage at D6 were included between January 2018 and May 2021. Two groups were compared : patients having a fresh D6 transfer (D6 fresh transfer group) and patients having a frozen thawed D6 transfer ( D6 frozen transfer group). Participants/materials, setting, methods 830 D6 single blastocyst transfers (736 frozen blastocysts and 94 fresh blastocysts) were analyzed. LBR and neonatal outcome were compared between groups, as well as the influence of clinical characteristics and data related to COS protocols. Correlation between D6 blastocyst morphology according to Gardner’s classification and livebirth occurrence was also evaluated. Statistical analysis of the data was carried out using univariate and multivariate logistic regression models. Main results and the role of chance LBR was significantly lower after D6 fresh blastocyst transfer compared to D6 frozen thawed blastocyst transfer [5.3% (5/96) vs 12.2% (90/736), p &amp;lt; 0.05]. Moreover, when comparing a subgroup of 68 first D6 frozen embryo transfers (1st D6 frozen transfer group) to the 94 fresh D6 blastocyst transfers (D6 fresh elective group), the superiority of D6 frozen blastocyst transfers regarding LBR was also confirmed (17.6% vs 5.3% p &amp;lt; 0.001, respectively). Concerning neonatal outcomes, the mean birth weight was comparable between the two groups. Univariate logistic regression analysis taking into account blastocyst morphology parameters showed that TE grade was the only parameter significantly associated with LBR after D6 embryo transfer (p &amp;lt; 0.001). Multiple logistic regression revealed that D6 embryo fresh transfer was independently associated with reduced LBR compared to frozen thawed D6 transfer (OR 0.367; 95%IC 0.143-0.945; p = 0.038). Moreover, our results showed that transferring a good or top quality D6 blastocyst increases 3 fold chances of live birth. Limitations, reasons for caution The number of fresh cycles seems relatively small, but the consistency of our results was guaranteed by the homogeneity of the 2 subgroups, standardization of all biological and clinical procedures and the robust statistical analysis methodology used in this study. Wider implications of the findings Despite their lower potential, D6 blastocysts have to be transferred with the objective to improve LBR, especially in women obtaining only these type of embryos. Our results recommend to transfer D6 blastocysts in frozen cycles. However, these findings have to be confirmed on larger series in prospective randomized trials. Trial registration number not applicable

P-701 Human embryos diagnosed as mosaic and aneuploid after preimplantation genetic testing for aneuploidy are associated with increased levels of apoptosis in trophectoderm cells

Abstract Study question Are there cell lineage-related differences in the apoptotic rates of human blastocysts classified as euploid, mosaic and aneuploid after preimplantation genetic testing for aneuploidy (PGT-A)? Summary answer In human blastocysts, apoptosis is more frequent in trophectoderm (TE) cells and is associated with the presence of mosaic and uniform chromosomal alterations. What is known already Embryos diagnosed as mosaic after PGT-A can develop into healthy infants. However, the reason why these embryos achieve reproductive competence needs further research. One hypothesis is that aneuploid cells are negatively selected during embryo development through cell competition mechanisms, such as apoptosis or differential proliferation. While these mechanisms have been demonstrated in mouse embryos, in which apoptosis occurs more frequently in the inner cell mass (ICM), evidence in human embryos is scarce. In fact, only one previous study has shown an association between mosaicism and aneuploidy after PGT-A and increased levels of apoptosis, which was more frequent in TE cells. Study design, size, duration Prospective cohort study performing colocalization of cell-lineage and apoptotic expression markers by immunofluorescence (IF). A total of 53 vitrified human blastocysts with a previous PGT-A diagnosis on day 5 (D5) or day 6 (D6) of development were analysed: n = 13 euploid embryos (n = 11 on D5; n = 2 on D6), n = 22 mosaic embryos (n = 16 on D5; n = 6 on D6), and n = 18 aneuploid embryos (n = 11 on D5; n = 7 on D6). All embryos were donated for research purposes. Participants/materials, setting, methods Following warming and re-expansion, blastocysts were fixed in 4% paraformaldehyde, processed for IF and imaged by confocal microscopy. Primary antibodies: 1:80 goat anti-human Nanog (AF1997, R&amp;D-Systems), 1:100 mouse anti-human Gata3 (MAB6330, R&amp;D-Systems) and 1:1000 rabbit anti-human Caspase-3 (NB10056113, Novus Biologicals). 165 μM DAPI was used for nuclei staining. Total cells: DAPI+ cells. TE: Gata3+ cells. ICM: Gata3-/Nanog+ cells. Incidence of apoptosis: % Caspase-3+ cells (per embryo). Data were analysed using Chi-square or ANOVA (p &amp;lt; 0.05 significant). Main results and the role of chance Total cell number was similar among euploid embryos (59.2±20.6 on D5; 78.5±0.7 on D6), mosaic embryos (49.6±15 on D5; 58.8±16.9 on D6) and aneuploid embryos (48.6±14.2 on D5; 59.6±23.5 on D6) (P &amp;gt; 0.05). Nanog and Gata3 expression evidenced the establishment of ICM and TE cell lineages during the blastocyst stage: while the proportion of Nanog+ cells decreased from 38.2% (755/1979) on D5 to 20.3% (188/927) on D6 (P &amp;lt; 0.0001), the proportion of Gata3+ cells increased from 77.8% (1540/1979) on D5 to 83.1% (770/927) on D6 (P &amp;lt; 0.01). The persistence of Nanog expression in TE cells was associated with the chromosomal status of the embryo, as evidenced by the significantly higher proportion of Gata3+/Nanog+ cells found in aneuploid embryos (27.9%=149/534 on D5; 4.6%=19/417 on D6) compared to mosaic embryos (13.1%=104/794 on D5; 3.4%=12/353 on D6) and euploid embryos (9.7%=63/651 on D5; 0%=0/157 on D6) (P &amp;lt; 0.05). The incidence of apoptosis in the TE was significantly higher in aneuploid embryos (45.9%±16.1 on D5; 49%±15.1 on D6) and mosaic embryos (44.1%±19.6 on D5; 43%±16.8 on D6), compared to euploid embryos (26.6%±16.6 on D5; 17.5%±14.8 on D6) (P &amp;lt; 0.05). Conversely, apoptosis was much less frequent in the ICM (2.2%±7.7), and no differences were found among the study groups (P &amp;gt; 0.05). Limitations, reasons for caution This is a descriptive, single-centre study with a limited sample size. The karyotype concordance between ICM and TE cells was not tested. Wider implications of the findings Our findings demonstrate that apoptosis is common in human blastocysts, particularly in TE cells. The increased apoptotic rates found in the TE of embryos with chromosomal abnormalities support its role as a mechanism to eliminate aneuploid cells. This mechanism may indeed be related to the reproductive potential of mosaic embryos. Trial registration number Not applicable

P-303 Outcomes of day 5 versus day 6 blastocysts: correlation of euploidy, implantation and embryo quality

Abstract Study question Are outcomes of day 5 (D5) blastocysts better than day 6 (D6) blastocysts in a PGT-A program? Summary answer Outcomes of day 5 embryos show more blastocysts, better quality, more euploidy due to better quality and higher implantation on same quality than day 6. What is known already Human embryos optimally reach the blastocyst stage after five days of culture, but some have a slower development. The superiority of day 5 blastocysts compared to day 6 blastocysts in fresh cycle transfers was previously demonstrated and attributed mainly to endometrial asynchrony. Data from frozen blastocysts transfers showed conflicting results, some studies have announced higher pregnancy rates after day 5 transfer compared with day 6 transfer, while others have shown equivalent outcomes. However, none of these studies have compared outcomes between same quality and euploid day 5 (D5) frozen blastocysts versus those frozen on day 6 (D6). Study design, size, duration Retrospective observational study performed in a private centre between May 2017 and December 2022. The study includes the data analysis of 5599 D5 and D6 blastocysts undergoing PGT-A obtained from 819 patients following 1295 PGS cycles. 948 euploid blastocysts with known implantation outcome were transferred in 789 frozen embryo transfers. Blastocyst morphology was scored in 3 groups: A: excellent (AA, AB, BA), B: good (BB), C: average and poor-quality embryos (BC, CB, CC). (Gardner-Schoolcraft classification) Participants/materials, setting, methods PGT-A with NGS technology was offered to patients of advanced maternal age and/or with repeated IVF failures. Trophectoderm biopsies were performed on D5 and/or D6 embryos, with laser assistance. (Navilase, OCTAX) We compared both populations (D5 and D6) in terms of number of blastocysts achieved and distribution of embryo quality of each population. As primary outcome, same-quality group results of euploid rate and implantation were compared according to the day of blastocyst development (D5 and D6). Main results and the role of chance Vast majority of the blastocysts were biopsied on D5 (71.8%, n = 4017) versus (28.2%, n = 1582) biopsied on D6. The proportion of three quality-categories of blastocysts according to D5/D6 is uneven. Quality A = 18.6% vs 3,5%. Quality B = 72,4% vs 59,6%. Quality C = 9,0% vs 36,9%, respectively, showing significative differences between D5/D6 (Chi^2 p-values of 3 groups&amp;lt; 0.001), concluding that overall D5 quality is better than D6. Overall euploidy rate on D5 population versus D6 was 31,3% vs 23,3% (p value &amp;lt;0.001), showing significant difference in favour of D5. But layering on 3 quality categories on D5/D6, the euploidy rate was 44,7% vs 51,8% for quality A, 29,6% vs 26,6% for quality B and 17,6% vs 14,9% for quality C, respectively, showing no significative differences D5/D6 (Chi^2 p-values: A = 0,331; B = 0,067; C = 0,280). Euploidy is not D5/D6 dependant but quality dependant. Overall implantation on D5 (65,9%) is higher than D6 (40,4%) (p value &amp;lt;0.001). Layering again, on D5 the implantation was 75,0% vs 66,7% for quality A, 63,8% vs 46,4% for quality B and 35,3% vs 26,3% for quality C, respectively. Despite implantation being higher on D5 on all three groups only group B shows D5/D6 significative differences. (Chi^2 p-values: A = 0,651; B &amp;lt; 0.001; C = 0,360). Implantation is embryo quality and D5/D6 dependant. Limitations, reasons for caution The study is limited by its retrospective nature and the low number of grade A D6-euploid blastocysts available to transfer. Additionally, it is common to transfer more than one grade C quality embryo to increase the chances of pregnancy, losing implantation track of these type. Wider implications of the findings It was uncontested that D5 blastocysts had better reproductive potential, but we managed to quantify this potential according to euploidy and implantation rates based on embryo quality, which remains the most important predictive biomarker for selecting the best embryo to transfer and reducing the time to achieve a pregnancy. Trial registration number not applicable

P-742 Universal genome-wide haplotyping-based preimplantation genetic testing: a systematic seven-year experience at a single center

Abstract Study question To record the outcome and clinical benefit of universal genome-wide haplotyping-based preimplantation genetic testing (PGT). Summary answer Universal PGT allows for standardization of protocols and reduces turnaround time. Apart from accurate genetic diagnosis, it also provides chromosome information, improving embryo selection strategies. What is known already Developments in genome-wide technologies leveraged the implementation of universal PGT that combines haplotyping and copy number typing across the whole genome. Universal PGT provides diagnosis for any familial monogenic disorder (PGT-M) or structural rearrangement (PGT-SR), and allows for concurrent aneuploidy screening (PGT-A) in PGT-M/SR cycles, all using the same generic protocol and the same biopsy. For this reason, various universal PGT approaches have been developed in the last 2-3 years and are increasingly making their way to the clinic worldwide. Study design, size, duration Universal PGT as a first-tier test was implemented at UZ Leuven mid-2014, using in-house developed algorithm (siCHILD/haplarithmisis). This is a retrospective analysis of universal PGT data, collected between 1 January 2015 and 31 December 2022. Data on PGT indication, PGT cycles and biopsy results was extracted. Implantation rate (IR; with IU/EU fetal sac) per embryo transfer was calculated from frozen embryo transfer (FET) cycles for patients with known result by the end of 2021. Participants/materials, setting, methods By the end of 2022, 718 couples have successfully finalized preclinical PGT workup. At the time of writing, 573 couples (80%) have further proceeded with PGT cycles. In total, 1264 PGT cycles were performed with day-3 (D3) cleavage-stage (n = 3344) or trophectoderm (TE) biopsies (n = 1524). For 403 patients with known outcome from 954 FET cycles, IR per embryo transfer was calculated per biopsy type and embryo ranking, considering genome-wide chromosome information. Main results and the role of chance Couples were referred to PGT due to various autosomal dominant (AD, 65%), autosomal recessive (AR, 14%) and X-linked disorders (XL, 10%). Various PGT-M indications encompassed &amp;gt;200 genes in total, but almost 20% of couples enrolled due to inherited BRCA1/BRCA2 mutations. PGT-SR was indication in 7% of cases, mainly for familial microduplications/deletions (64%). For all PGT cycles, approximately 15% and 18% of all performed D3 and TE biopsies, respectively, were scored as abnormal largely due to meiotic errors in the embryo. All unaffected embryos were further ranked as R1 (euploid) or R2 (mosaic), and the number of R2 embryos was expectedly higher after D3 biopsies, compared to TE biopsies (250/1063, 23.6% vs 37/510, 7.3%, p&amp;lt;0.001). In accordance with inheritance mode, the number of unaffected embryos suitable for transfer was significantly lower in AD PGT cycles, compared to AR (32.3% (1116/3455) vs 44.0% (262/596) ; p&amp;lt;0.0001)). For 403 patients with FET cycles, IR was higher in R1 vs R2 embryos after D3 biopsy (231/590, 39.2% vs 30/159, 18.9 %, p &amp;lt;0.0001), whereas no difference was observed between R1 and R2 embryos after TE biopsy, although low number of R2 embryos after TE biopsy was transferred (85/142, 59.9% vs 7/12, 58.3%, p=ns). Limitations, reasons for caution The analyzed data is derived from a single center and the clinical outcome may not reflect the experience of other IVF/PGT centers due to confounding factors, such as PGT indication, used PGT technology and IVF lab practice. Wider implications of the findings The current dataset provides a comprehensive overview on general trends and outcome of universal PGT practice. Apart from accurate genetic diagnosis, universal PGT provides information on chromosomes, which improves embryo selection for transfer. This in turn impacts clinical counselling, interpretation and reporting of the results, and embryo transfer policies. Trial registration number Not applicable

O-096 Effect of Day-3 embryo morphology on blastocyst quality and ploidy: a machine-learning model of 34781 embryos

Abstract Study question What is the predictive value of using Day-3 (D3) embryo morphology and clinical factors to identify good quality euploid blastocysts? Summary answer Using a machine-learning model, D3-cell count and fragmentation helps identifying embryos most and least likely to develop into euploid blastocysts. What is known already It is well-known that the pattern of embryo cleavage affects blastocyst ploidy. Less has been elucidated in relation to embryo fragmentation. Published studies often used outdated biopsy techniques (D3-biopsy) and obsolete genetic technologies (FISH). Hence, the association between embryo fragmentation and ploidy status remains controversial. Existing hypothesis is that embryo fragmentation is a regulation event to maintain homeostasis and normalize genetic constitution. Literature is lacking studies investigating this association using newer genetic technologies such as Next Generating Sequencing (NGS). Moreover, a predictive model using D3-embryo characteristics and clinical factors can be useful for triage of laboratory resources. Study design, size, duration Retrospective observational single center study including 34781 embryos from 5701 cycles, between March 2017 and March 2020. Embryos were evaluated for degree of fragmentation and cell number on D3, 68±1hrs post insemination. Female Age, AMH and BMI were annotated as patient characteristics. All blastocysts available on D5 or D6 with a quality ≥ BL3CC (n = 18361) were subjected to trophectoderm (TE) biopsy for Preimplantation-Genetic-Testing for aneuploidies (PGT-A) analysis with Next-Generation-Sequencing and ploidy rates were recorded. Participants/materials, setting, methods Degree of fragmentation was classified as follows: A( &amp;lt; =10%), B(11-25%), C(26-35%), D(&amp;gt;35%). Data on embryo fragmentation was stratified according to cell number category: &amp;lt;6cells, 6-10 cells, &amp;gt;10 cells and compacted stage. Morphology markers (cell-count and fragmentation rate), along with patient characteristics (age, anti-Mullerian hormone and body-mass index) was modeled using a gradient-boosting decision tree algorithm. Performance of the machine learning (ML) model was assessed with AUC values, calibration curves after partitioning the dataset 1:1 for training/validation. Main results and the role of chance Day-3 embryos consisting of &amp;lt; 6 cells (n = 5926), 6-10 cells (n = 23920), and &amp;gt;10 cells (n = 1612) were included, along with 3323 compacted embryos. The rate of good-quality blastocysts decreased with higher fragmentation rates (38.2%, 14.8%, 4.6%, and 1.6%, A, B, C, and D, respectively; P &amp;lt; 0.001). Day of biopsy was earlier for embryos with lower fragmentation rates (Day-5 biopsy: 24.2%, 9.7%, 4.0%, and 0.0%, P &amp;lt; 0.001). Among the biopsied blastocysts, euploidy rates were similar at 44.9%, 43.0%, 42.7%, and 66.7% in groups A vs B, C, and D, respectively (P = 0.075, 0.350 and 0.141). When stratified by cell number, euploidy rates were lowest in blastocysts derived from &amp;lt;6-cells, at 33.5%, 34.3%, 26.9%, and 50.0% in groups A vs B, C, and D, respectively (P = 0.803, 0.185 and 0.394). The best performance ML model was provided by using fragmentation % and cell count along with female age, BMI and AMH. This resulted in a modest predictive accuracy for predicting euploid embryo development (AUC: 0.72, 95% CI: 0.71 to 0.73). The model was well- calibrated and correctly assessed the probability of D3-embryos developing into euploid blastocyst. For instance, embryos with a predicted probability of ≤ 2.5% rarely developed into euploid blastocyst (actual rate: 2.6%) and model had a predictive value of 97.4%.Limitations, reasons for caution: The retrospective nature of the study and inter-observer variability in D3-embryo fragmentation scoring is a limitation. Nevertheless, embryologists performing the embryo scoring followed the same SOPs. Findings need to be validated in external cohorts. Wider implications of the findings The model can utilize clinical factors and D3-embryo morphology markers to estimate the probability of a D3-embryo developing into an euploid blastocyst. This model can be useful for patient information, mainly when a decision on embryo selection for PGT-A needs to take place. Trial registration number not applicable

O-024 Exploring the optimal transfer day of day 6 (D6) vitrified blastocysts in Hormone Replacement Therapy Frozen Embryo Transfer (HRT-FET) cycles - a cohort study

Abstract Study question Should blastocysts cryopreserved D6 be transferred on the 5th or 6th day of progesterone administration in HRT-FET cycles? Summary answer No significant differences in outcome parameters were found when D6 vitrified blastocysts were transferred on either day 5 or day 6 of progesterone in HRT-FET. What is known already Several studies have shown that D6 blastocyst transfer should be avoided in fresh transfer IVF cycles; consequently, these more slowly developing embryos are transferred in a HRT-FET cycle on the 6th day of progesterone administration in line with day 5 (D5) blastocysts. For successful implantation, a receptive endometrium and optimal synchronization between the endometrium and embryo are important. However, no definitive evidence exists regarding the optimal timing of transferring D6 blastocysts. Interestingly, a few studies have reported better reproductive outcomes if D6 blastocysts are transferred on the 5th day of progesterone administration instead of the 6th day of progesterone administration. Study design, size, duration This cohort study included a total of 718 vitrified D6 single blastocyst HRT-FET cycles performed between 2013 and 2022. 576 blastocysts were transferred on the 6th day of progesterone administration (2013-2021), and 142 blastocysts transferred on the 5th day of progesterone administration (2021-2022). Main outcomes were biochemical pregnancy rate (PR), implantation rate (IR), clinical pregnancy rate (CPR), and early pregnancy loss rate (EPLR). Participants/materials, setting, methods Patients undergoing HRT-FET in a public Fertility Clinic. Endometrial preparation included oral oestradiol (6mg/24hours), followed by vaginal micronized progesterone. As mentioned above, single blastocyst transfer was performed on either the 5th or 6th day of progesterone administration. Serum β-hCG level was measured 9-11 days after embryo transfer and biochemical pregnancy was defined as an hCG level &amp;gt;10 IU/L. Clinical pregnancy was defined as visualization of a gestational sac and fetal heartbeat in gestational week 7. Main results and the role of chance In this cohort of a total of 718 HRT-FET cycles transferred on either the 5th or 6th day of progesterone administration the mean age of the patients at embryo transfer was 34.5±4.5 years vs. 33.0±4.9 years, respectively, p = 0.30. There was no significant difference between numbers of top-quality blastocyst transfers within the two groups 63% (89/142) vs. 64% (369/576), p = 0.76. The overall, biochemical pregnancy rate (PR), implantation rate (IR), clinical pregnancy rate (CPR) and early pregnancy loss (EPLR) in the group of patients transferred on the 5th day of progesterone administration were 38% (54/142), 29% (41/142), 25% (36/142) and 33% (18/54). In the group of patients transferred on the 6th day of progesterone administration 41% (235/576), 33% (191/576), 30% (170/576) and 28% (65/235). There were no significant differences between PR, IR, CPR and EPLR when transferring D6 vitrified blastocysts on either the 5th or the 6th day of progesterone administration (38% vs. 41%, p = 0.53, 29% vs. 33%, p = 0.33, 25% vs. 30%, p = 0.33 and 33% vs. 28%, p = 0.41). Limitations, reasons for caution Limitations of the study are the retrospective design and the limited number of D6 vitrified blastocysts transferred on the 5th day of progesterone administration. Furthermore, the two groups represented two different time periods. Wider implications of the findings Growing evidence shows that D5 vitrified blastocysts have better reproductive outcomes than slower developing embryos which are vitrified on day 6. Whether this is caused by intrinsic embryonic factors or poor timing of transfer in a subsequent FET cycle still needs to be unraveled. Trial registration number not applicable

The effectiveness of a health education intervention to reduce anxiety in quarantined COVID-19 patients: a randomized controlled trial

IntroductionThe COVID-19 pandemic is regarded as a serious public health concern that boosts levels of stress and anxiety which could be explained by several reasons, including social isolation. In this regard, we aimed to assess the impact of health education on the anxiety level of COVID-19 patients during the isolation period.MethodsThis is a randomized controlled trial conducted between February 2021 and June 2021. Patients tested positive for Covid-19 with mild to moderate forms were randomized to Education (n = 267) or control (n = 269). The education group received a phone health education session on day 1 (D1) following the diagnosis. The three components of the health education intervention were an explanation of the coronavirus disease, what to do in the event of complications, and the recommended preventive measures. The two groups received a telephone evaluation of their Hospital Anxiety and Depression scores on D1 and day seven D7 following the positive diagnosis. The primary outcome was the rate of anxiety reduction in each group on D7 based on a HAD-A score ≥ 8. Secondary outcomes were the rate of anxiety reduction on D7 based on a HAD-A score ≥ 11, the percentage of people complying with isolation and the scores of adherences to preventive measures during the isolation in each group.ResultsHundred and ninety-six patients in the intervention group and 206 patients in the control group completed the study. The sociodemographic, clinical, and initial anxiety level features of the intervention and control groups were comparable at baseline (p ≥ 0.05). On D7, the education group’s anxiety level (HAD-A ≥ 8) decreased from 26 to 16.3% (p = 0.013) while in the control group it increased from 19.4 to 22.8% (p = 0.37). Thus, the percentage change in anxiety between D1 and D7 (delta D7 – D1) was − 9.7% in the Education group and + 3.4% in the Control group. Using the HAD-A ≥ 11 thresholds, the percentage of anxiety decreased from 15.3 to 11.2% (p = 0.26) between D1 and D7, while it increased in the control group from 9.7 to 15.7% (p = 0.045). Thus, the education group’s change in anxiety (delta D7 - D1) was − 4.1%, while the control group’s change was + 6%.ConclusionDuring an outbreak, providing health education to quarantined patients may be beneficial to reduce the psychological impact of the disease.Trial registration numberClinicalTrials.gov Identifier: NCT05715593, retrospectively registered on 8/02/2023 https://clinicaltrials.gov/ct2/results?term=NCT05715593&Search=Search.