Short Protocol Research Articles

P-603 Presence of an asymmetrical response to ovarian stimulation in patients with low ovarian reserve

Abstract Study question Does ovarian reserve decline with a symmetrical manner between right and left ovaries in poor responders (POR) with diminished ovarian reserve (DOR)? Summary answer Asymmetrical ovarian response to ovarian stimulation with the left-side dominance was found in POR with DOR. What is known already Ovarian follicles are produced during fetal stage and not regenerated after birth. Thus, the number of ovarian follicles declines with age, resulting in infertile POR with DOR. In the morphometric study of human neonatal ovaries, no significant difference was found in the number of follicles between the right and left ovaries in the same individual. A previous study demonstrated that there is a difference in the number of follicles between right and left ovaries in patients with normal ovarian reserve with the right-side dominance, suggesting the asymmetrical activation and growth of follicles. Study design, size, duration A retrospective analysis was conducted in patients with POR with DOR based on the Bologna Criteria. Inclusion criteria was patients who received more than five times of ovarian stimulations followed by oocyte retrievals. Data were obtained from a total of 265 participants who received IVF-ET treatments from April 2015 to March 2021 after receiving written informed consents under an approval from the institutional ethical committee. Patients with the history of previous ovarian surgery were excluded. Participants/materials, setting, methods The enrolled patients were received ovarian simulation under short or GnRH antagonist protocols for oocyte retrieval. We collected the data of retrieved oocyte number as well as the outcome of IVF from medical chart. We defined the right-left asymmetry of ovarian reserve (%) based on the number of retrieved oocytes from dominant side ovary per total number of retrieved oocytes. Statistical significance was determined using Dunnett or chi-square tests, with P < 0.05 being statistically significant. Main results and the role of chance The average age of participants was 37.2±5.99 years of age exhibiting low serum AMH levels (average 0.09±0.20 ng/ml). We analyzed 2,181 cycles of ovarian stimulation (average 8.3±3.9 cycles/patient). The number of retrieved oocytes were 3, 882 in total cycles (average 12.8±7.1/patient). Among participants, 22 cases (8.4%) showed left and right equal in the number of retrieved oocytes, whereas >70% asymmetry was observed in 107 cases (40.7%) and >80% asymmetry was detected in 60 cases (22.8%). In 18 cases (6.9%), oocytes were collected from one side ovary only showing 100% asymmetry. In the cases with >70 and 100% asymmetry, the left-side dominance was 1.3-fold and 5.0-fold higher than right-side dominance, respectively. In cases with 100% asymmetry, there was no difference in the number of cryopreserved high-quality embryos between left and right sides of ovary. Limitations, reasons for caution Although we enrolled POR with DOR patients who received ovarian stimulations more than five times, the duration of ovarian stimulation was different among patients. It affects the numbers of ovarian stimulation cycles and retrieved oocytes in each patient. Wider implications of the findings Considering the finding of right-side dominance in the number of follicles with normal ovarian reserve, the activation and development of follicles might be accelerated in the right side due to asymmetric blood supply to the ovaries, and thus follicles are likely remained in the left-side ovary with low ovarian reserve. Trial registration number not applicable

P-673 GnRH Agonist Early Follicular Challenge Test as a Predictor of Ovarian Response in Short Antagonist Cycles for Fertility Preservation

Abstract Study question Does the response to GnRH agonist can be used to predict ovarian function in short GnRH antagonist cycles in women undergoing oocyte cryopreservation IVF cycles? Summary answer Response to a single GnRH agonist dose at the beginning of stimulation during short antagonist cycle can predict the outcome of the fertility preservation cycle What is known already There is a need for a reliable test for ovarian reserve. Gonadotrophin agonist stimulation test (GAST) is considered a valid marker for ovarian reserve but the test was never validated for short antagonist protocol. The reported dose for this test was 0.1 mg, however, from the vast experience with GnRH agonist triggering, it is now agreed that the optimal dose for GnRH agonist flare effect is 0.2 mg. Study design, size, duration A prospective observational study of fertility preservation cycles patient in an academic hospital setting. Seventy-four Short GnRH antagonist cycles that underwent an oocyte retrieval between 1 December 2020 and 1 January 2022. Participants/materials, setting, methods On day 2, blood was sampled for basal FSH, LH, and E2 measurements, followed by a subcutaneous injection of 0.2mg GnRH agonist as part of the initial ovarian stimulation. Twelve hours later blood sampling was repeated. E2 response was used as test parameter. The major outcome was the number of cryopreserved oocytes. Main results and the role of chance Participants were divided to groups according to Day 3 E2 to Day 2 E2 response (E3/E2 ratio) to lower and upper third percentiles (<3.32, n = 24 and >5.41, n = 25). Age (32.68 vs. 32.79, p = 0.965), total gonadotropin dose injected 3216.67 vs. 2773.42 IU p = 0.16) and day 3 FSH levels (7.39 vs. 7.16, p = 0.768) were not significantly different between groups. Ovarian response as measured by E2 levels on ovulation trigger (7879.72 vs. 17572.54 pmol/ml, p = 0.001) and number of M2 oocytes retrieved were significantly different (17.92 vs. 6.24, p < 0.001). Linear correlation between E3/E2 ratio and number of M2 oocytes was calculated (R = 0.48, p < 0.001). ROC curve analysis of E3/E2 ratio for more than fifteen M2 oocytes indicate AUC value of 0.82 (cutoff value of 4.22, p < 0.001, 84.6% sensitivity, 70.8% specificity) and for less than five M2 oocytes AUC value of 0.84 (cutoff value of 3.34, p < 0.001, 76.3% sensitivity, 73.3% specificity). The results suggest that the testing response to 0.2mg GnRH agonist positively correlates with treatment outcome of the current cycle. Limitations, reasons for caution Although all patients were tested for FSH levels for ovarian reserve, they were not tested for AMH levels. Wider implications of the findings The response to single GnRH agonist dose during short antagonist cycle can be used as another biomarker of ovarian reserve. This simple, widely available marker, which reflect the estradiol response of small follicles, might predict accurately the outcome of the specific cycle, and potentially used to adjust the treatment dose. Trial registration number NCT04973969

P-570 Prokineticin-1 follicular fluid levels are strongly associated with diminished ovarian reserve and poor ovarian response: A prospective observational study

Abstract Study question Are Prokineticin-1 (PROK1) follicular fluid (FF) levels associated with poor ovarian response (POR) as well as with other well-known biomarkers associated with ovarian reserve status? Summary answer Poor responders present with increased PROK1 FF levels. Also, PROK1 FF levels are negatively correlated with anti-müllerian hormone (AMH) levels and number of oocytes retrieved. What is known already Prokineticin-1 is an angiogenic factor with pleiotropic properties. Considering its role in reproduction, PROK1 mediates significant angiogenic functions in the fetal-maternal interface. Thus, alterations in PROK1 expression and/or function lead to placenta-derived pregnancy complications. Recently published studies also indicate that PROK1 is associated with ovarian function and oocyte competence. However, limited data associating PROK1 with POR are published. This study uniquely investigates possible associations between PROK1 FF levels in POR cases, with stimulation outcome, as well as with FF levels of other factors related to ovarian functionality, namely VEGF, bone morphogenetic protein 15 (BMP-15) and Pigment Epithelium Derived Factor (PEDF). Study design, size, duration This prospective-observational study was collaboratively conducted between November 2020 and July 2021 at Genesis Athens Clinic and at the University of Athens Medical School. A total of 64 patients undergoing IVF treatment were enrolled. The study group comprised of 32 POR patients defined according to the Bologna criteria. The control group consisted of 32 normal responder women undergoing IVF due to tubal factor and/or mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. Prior to ovarian stimulation, participants were subjected to basic infertility investigation, including antral follicle count and AMH levels evaluation. The FF samples were collected as part of the oocyte retrieval process, then centrifuged and stored at -80 °C till analysis. Follicular fluid levels of PROK1, VEGF, BMP-15 and PEDF were evaluated via ELISA employing commercially available kits. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Significantly higher PROK1 (3229.63 ± 2372.66 vs 1945.95 ± 1408.01 pg/ml; P-value<0.0001), VEGF (2309.63 ± 412.49 vs 2013.22 ± 330.16 pg/ml; P-value=0.006) and lower BMP-15 (434.13 ± 106.38 vs 532.5 ± 108.26 pg/ml; P-value=0.001) levels were recorded in the POR group. No difference was observed regarding PEDF levels (7.43 ± 1.95 vs 8.23 ± 2.54 ng/ml; P-value=0.2). However, the POR group presented with lower PEDF/VEGF ratio (3.33 ± 1.08 vs 4.18 ± 1.41; P-value=0.02), indicating a reduced antioxidant capacity. PROK1 levels were negatively correlated with AMH (P-value=0.04), number of oocytes retrieved (P-value=0.001) and number of MII oocytes (P-value=0.005). BMP-15 was positively correlated with number of oocytes retrieved (P-value<0.001), number of MII oocytes (P-value=0.002), number of normally fertilized zygotes (P-value=0.007), number of cleavage stage embryos (P-value=0.03) and number of blastocysts (P-value=0.04). VEGF was negatively correlated with AMH (P-value=0.004) and number of oocytes retrieved (P-value=0.03). These correlations remained significant when adjusting for ovarian response status. PROK1 levels, with a cut-off value of 2854.25 pg/ml, were able to predict ovarian response status with an area under the curve at 0.64. Sensitivity was 0.55, specificity was 0.88, and accuracy was 0.71. The positive and negative predictive values were 81.82% and 66.67%, respectively. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data considering pregnancy outcomes. Moreover, molecular data with regards to the possible role of PROK1 on POR pathogenesis are required. Future studies are needed to verify the results presented herein. Wider implications of the findings Data presented herein indicate that PROK1 FF levels are strongly associated with diminished ovarian reserve and POR. Considering that FF-PROK1 presents with a similar profile with FF-VEGF, we can form the hypothesis that the compromised angiogenesis observed in POR patients leads to PROK1 and VEGF increase via a negative-feedback-loop. Trial registration number Not applicable

O-139 COVID-19 in pregnant women: a living systematic review and meta-analysis on the risk and prevalence of pregnancy loss

Study questionWhat is the risk and prevalence of pregnancy loss (PL) in women with COVID-19 compared to women without COVID-19?Summary answerPregnant women with COVID-19 do not appear to be at increased risk of miscarriage.What is known alreadyPregnant women with COVID-19 have an increased risk to deliver preterm and to deliver a stillborn child in comparison to pregnant women without the disease. Currently, many studies have evaluated birth outcomes in pregnant women with COVID-19, however few regard the risk of PL as most data were available on pregnancies infected during the third trimester. Based on the data currently available, there is no evidence to suggest that an infection with SARS-CoV-2 poses an increased risk of miscarriage.Study design, size, durationOur living systematic review is based on a prospectively registered protocol (PROSPERO CRD42020178076; registered 22 April 2020). For this project a short separate protocol was developed (https://osf.io/e8dhr/). The literature search was performed up until the 8th of December 2021 and an update has been planned.Participants/materials, setting, methodsWe included retrospective and prospective cohort studies of pregnant women with COVID-19, provided that they contained information on PL. We calculated odds ratios (OR) and risk differences with corresponding 95% confidence intervals (CI) and pooled the data using random effects meta-analysis. To estimate risk prevalence, we performed meta-analysis on proportions. Heterogeneity was assessed by I2.Main results and the role of chanceWe included 75 studies comprising a total of 39826 pregnant women, of which 32663 pregnant women with COVID-19 and 2610 controls. 1423 pregnant women with COVID-19 were in their first trimester and 2700 were in their second trimester. Evidence level was considered to be of low certainty. The overall proportion of pregnancy loss in all COVID-19 pregnancies including third trimester pregnancies was 1.2% (95% CI 0.7% to 1.8%; I2=79%). Selecting on first and second trimester pregnancies the proportion of pregnancy loss was 4.7% (95% CI 3.0% to 6.7%; I2=71%).When comparing pregnancy loss in cohorts of pregnant women with COVID-19 to pregnant women without the disease the odds ratio was 1.26 (95% CI 0.74 to 2.18, I2=0%; RD 0.12%, 95% CI -0.62 to 0.85, I2=0%). Selecting on only first and second trimester pregnancies, the odds ratio for pregnancy loss was 1.35 (95% CI 0.46 to 3.93, I2=0%; RD 0.35%, 95% CI -1.44 to 2.15, I2=0%;).The overall proportion of ectopic pregnancy in all COVID-19 pregnancies cohorts including third trimester pregnancies was 0.61% (95% CI 0.1% to 1.4%; I2=49.8%). Selecting on first and second trimester pregnancies the proportion of ectopic pregnancy was 2.0% (95% CI 0.3% to 5.9%; I2=40%).Limitations, reasons for cautionMost included studies were hospital-based studies such that selection bias towards more severe infections seems likely. A minority of studies reported on first and second trimester pregnancies.Wider implications of the findingsAt this moment there are no indications that COVID-19 increases the risk of miscarriages. In view of the wide insecurities around the risk estimates further well-designed studies are required, that consider the clinical manifestation of COVID-19 and include first and second trimester pregnancies.Trial registration numberNot applicable

P-571 The newly developed highly sensitive reagent GL13 indicates extensively increased cellular senescence in the follicular fluid of poor responder patients: A prospective observational study

Abstract Study question Do women of diminished ovarian reserve and poor ovarian response (POR) present with increased lipofuscin follicular fluid (FF) levels, indicating increased ovarian cellular senescence? Summary answer Poor responders present with a six-fold increased lipofuscin FF levels indicating extensive senescence in POR ovaries. Lipofuscin may be a new sensitive biomarker for POR. What is known already Lipofuscin is a nondegradable substrate of metabolism accumulating in cells due to impaired mitochondrial/lysosome/proteasome function, upon stress or damage. It is well-documented that lipofuscin constitutes a highly sensitive biomarker of cellular aging and senescence. Recently, a novel reagent (GL13) coupled by a hybrid histochemical–immunohistochemical method were developed for detecting and measuring soluble lipofuscin levels in biological fluids, ascertaining high sensitivity and specificity. Data indicate that lipofuscin levels are associated with oocyte competence and age-related infertility. However, hitherto no data has been published indicating the value of lipofuscin FF levels as a biomarker towards accurately predicting ovarian reserve and response. Study design, size, duration This prospective observational study was collaboratively conducted between November 2020 and September 2021 at the Athens University Medical School and at Genesis Athens Clinic. A total of 32 patients undergoing IVF treatment were enrolled. The study group comprised of 16 POR patients defined according to the Bologna criteria. The control group consisted of 16 normal responder women undergoing IVF due to tubal factor or/and mild male factor infertility. Patients with other infertility aetiologies were excluded. Participants/materials, setting, methods Participants in both groups received the standard short GnRH-antagonist protocol. The FF samples were collected as part of the oocyte retrieval process. Lipid and protein parts of lipofuscin were isolated from FF samples and stained with GL13. The complex of lipofuscin-GL13 was labeled with an anti-biotin HRP conjugated antibody and detected employing a chemiluminescence reaction. Luminescence was measured and signal intensity was corresponding to lipofuscin concentration. Statistical analysis was performed employing R Programming Language. Main results and the role of chance Poor responders presented with a statistically significant six-fold higher lipofuscin FF levels in comparison to the normal responder group (869.21 ± 501.87 vs 146.6 ± 107.64 RLU; P-value <0.0001). Lipofuscin levels were negatively correlated with AFC (Spearman’s Rho: -0.68; P-value <0.0001), AMH levels (Rho: -0.61; P-value =0.0002), estradiol levels on triggering day (Rho: -0.68; P-value <0.0001), number of oocytes retrieved (Rho: -0.54; P-value =0.001), number of mature metaphase II (MII) oocytes obtained (Rho: -0.58; P-value =0.0008), number of normally fertilized (2PN) zygotes (Rho: -0.51; P-value =0.003), number of cleavage stage embryos (Rho: -0.48; P-value =0.005) and number of blastocyst stage embryos (Rho: -0.41; P-value =0.02). These correlations remained statistically significant when adjusting for ovarian stimulation response status. No association was established between Lipofuscin levels and embryo quality neither on Day 3 nor on Day 5. Lipofuscin levels, with a cut-off value at 294, were able to predict ovarian stimulation response status with an area under the curve (AUC) at 0.96. The sensitivity was 0.875, the specificity was 0.938 and the accuracy was 0.906. The positive predictive value was 88.24% and the negative predictive value was 93.33%. Limitations, reasons for caution Limitations of our study refer to the limited size of the studied population, as well as to the lack of data referring to pregnancy outcomes. Moreover, molecular data with regards to the possible mechanisms leading to the observed increased senescence are required. Future studies are needed to verify these findings. Wider implications of the findings Data presented herein indicates, for the first time in literature, that lipofuscin FF levels measured via the GL13 method may be a promising and sensitive tool for predicting POR and stimulation outcome. Lipofuscin could further serve as a valuable novel biomarker indicating ovarian senescence, ovarian reserve status and oocyte competence. Trial registration number Not Applicable

P-158 Beneficial effects of platelet rich plasma for human germinal vesicle oocyte in vitro maturation

Abstract Study question Can platelet rich plasma (PRP) supplementation to the medium improve outcomes of in vitro maturation of germinal vesicle (GV) oocytes? Summary answer PRP as a component of in vitro maturation medium benefits the GV oocyte maturation rate, the mitochondrial membrane potential and the distribution of lipid droplets. What is known already The European Society of Human Reproduction and Embryology presents several strategies for maintaining fertility in women, one of which is in vitro maturation (IVM) of oocytes. Despite more than 5,000 children have been born after using the IVM method, the mechanisms involved in meiotic arrest and resumption in oocytes are not fully understood. Hormones and growth factors are components of a complex regulation system which play a mediator role in ovarian function and are involved in meiotic resumption. PRP is a rich source of growth factors and can be effectively used to supplement the medium for in vitro oocyte maturation. Study design, size, duration Oocyte maturity was assessed after denudation, and immature gametes at the GV stage (n = 84) were randomly divided into 4 groups for IVM (24 - 48h): 1- without cumulus cells (CC) and PRP, 2 – with PRP without CC, 3 - with CC without PRP, 4 – with CC and PRP. The maturation rate of the oocytes was evaluated in 24 or 48h. The study was carried out between January 2020 to December 2021. Participants/materials, setting, methods This prospective study included 30 female patients 33±3.6 years old. Women received exogenous gonadotropins as part of a short protocol of superovulation induction. About 15% oocytes were at the germinal vesicle stage after denudation of retrieved gametes. PRP was prepared by two-step centrifugation method and was added (5%) to the maturation media. Primary endpoints were oocyte maturation rate, mitochondrial membrane potential and lipid droplets distribution evaluated by using JC-1 and Nile Red staining respectively. Main results and the role of chance The highest maturation rate was in group 4 (85%) compared with groups 1-3, where the rates were 9.5; 36.4 and 47.6, respectively (p < 0.05). The biggest number of cells with high mitochondrial potential was in oocytes matured with PRP and cumulus cells (4.5 ± 0.7) compared with other groups (1.3 ± 0.3; 3.0 ± 0.5 and 2.7 ± 0.4 for groups 1-3, respectively) (p < 0.05). The distribution of lipid droplets, typical of in vivo matured metaphase II oocytes, was the largest in group 1 (75%) (p < 0.05). There were no differences in this parameter between the other groups. Limitations, reasons for caution Limited number of GV oocytes in the study group. Further studies are recommended and should include group of patients with different age range. Wider implications of the findings PRP injection into ovarian tissue causes its rejuvenation. Our results confirm for the first time that PRP supplementation to the medium of oocyte in vitro maturation can increase the maturation rate, mitochondrial activity, and improve lipid droplets distribution in human oocytes. These finding are valuable for improvement of IVM medium. Trial registration number Not applicable

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy.

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models.

A Case Study in Meaningful Assessment of an Interdisciplinary Cultural Competency Graduation Requirement

In an institution where assessment was viewed as meaningless “box checking” and for the purposes of accreditation only, a new reflective assessment practice with 15 faculty changed their perception of assessment in a single semester. Piloted with an interdisciplinary graduation requirement of Cultural Competency, the new assessment practice became a powerful tool for “closing the loop” in which participating faculty analyzed evidence to make improvements for student learning. This article is a detailed history of the inception and explanation of that practice and the evidence of the faculty appreciation for the new four question short form protocol.

Severity Level and Duration of Energy Deficit in Mice Affect Bone Phenotype and Bone Marrow Stromal Cell Differentiation Capacity.

Anorexia nervosa is known to induce changes in bone parameters and an increase in bone marrow adiposity (BMA) that depend on the duration and seriousness of the disease. Previous studies have found that bone loss is associated with BMA accumulation. Sirtuin of type 1 (Sirt1), a histone deacetylase that is partly regulated by energy balance, was shown to have pro-osteoblastogenic and anti-adipogenic effects. To study the effects of the severity and duration of energy deficits related to bone loss, a mouse model of separation-based anorexia (SBA) was established. We recently demonstrated that moderate body weight loss (18%) 8-week SBA protocol in mice resulted in an increase in BMA, bone loss, and a significant reduction in Sirt1 expression in bone marrow stromal cells (BMSCs) extracted from SBA mice. We hypothesised that Sirt1 deficit in BMSCs is associated with bone and BMA alterations and could potentially depend on the severity of weight loss and the length of SBA protocol. We studied bone parameters, BMA, BMSC differentiation capacity, and Sirt1 expression after induction of 4 different levels of body weight loss (0%,12%,18%,24%), after 4 or 10 weeks of the SBA protocol. Our results demonstrated that 10 week SBA protocols associated with body weight loss (12%, 18%, 24%) induced a significant decrease in bone parameters without any increase in BMA. BMSCs extracted from 12% and 18% SBA groups showed a significant decrease in Sirt1 mRNA levels before and after co-differentiation. For these two groups, decrease in Sirt1 was associated with a significant increase in the mRNA level of adipogenic markers and a reduction of osteoblastogenesis. Inducing an 18% body weight loss, we tested a short SBA protocol (4-week). We demonstrated that a 4-week SBA protocol caused a significant decrease in Tb.Th only, without change in other bone parameters, BMA, Sirt1 expression, or differentiation capacity of BMSCs. In conclusion, this study showed, for the first time, that the duration and severity of energy deficits are critical for changes in bone parameters, BMSC differentiation, and Sirt1 expression. Furthermore, we showed that in this context, Sirt1 expression could impact BMSC differentiation with further effects on bone phenotype.

Cohort profile: Anhui Maternal–Child Health Study in China

PurposeThe Anhui Maternal–Child Health Study (AMCHS) aims to examine determinants of reproduction, pregnancy and postpartum maternal and child health outcomes in Chinese women who received assisted reproductive technology (ART).Study design...

Production and Characterization of Rat Monoclonal Antibodies against the PAL Antigen of Legionella spp.

The purpose of this work was to obtain genus-specific monoclonal antibodies against the Legionella spp. recombinant PAL protein, which will subsequently allow to use them as a basis for the development of new express tests for pathogenic legionella detection. A short three-week immunization protocol for Wistar rats was used to generate rat–mouse heterohybridomas producing antibodies against PAL. Mouse myeloma cell line Sp2/0-Ag14 served as the fusion partner. Hybridization was performed using two methods: PEG-mediated fusion and electrofusion. Subsequent screening was performed by indirect solid-phase ELISA against the target protein rPAL. Specificity analysis was performed by dot-blot using a panel of lysates obtained from 39 pure cultures of different strains, which included closely related and heterologous microorganisms among others. No difference in the efficiency of stable hybridoma clones production by the two indicated cell-fusion methods was detected. Twelve clones producing specific rat monoclonal antibodies were obtained based on the screening results. The obtained rat monoclonal antibodies are highly specific towards the PAL protein of L. pneumophila of different serological groups and other pathogenic legionella and are good candidates to be used as the components of diagnostic test systems for the detection of pathogenic representatives of the Legionella genus.

Stance Phase Gait Training Post Stroke Using Simultaneous Transcranial Direct Current Stimulation and Motor Learning-Based Virtual Reality-Assisted Therapy: Protocol Development and Initial Testing.

Gait deficits are often persistent after stroke, and current rehabilitation methods do not restore normal gait for everyone. Targeted methods of focused gait therapy that meet the individual needs of each stroke survivor are needed. Our objective was to develop and test a combination protocol of simultaneous brain stimulation and focused stance phase training for people with chronic stroke (>6 months). We combined Transcranial Direct Current Stimulation (tDCS) with targeted stance phase therapy using Virtual Reality (VR)-assisted treadmill training and overground practice. The training was guided by motor learning principles. Five users (>6 months post-stroke with stance phase gait deficits) completed 10 treatment sessions. Each session began with 30 min of VR-assisted treadmill training designed to apply motor learning (ML)-based stance phase targeted practice. During the first 15 min of the treadmill training, bihemispheric tDCS was simultaneously delivered. Immediately after, users completed 30 min of overground (ML)-based gait training. The outcomes included the feasibility of protocol administration, gait speed, Timed Up and Go (TUG), Functional Gait Assessment (FGA), paretic limb stance phase control capability, and the Fugl–Meyer for lower extremity coordination (FMLE). The changes in the outcome measures (except the assessments of stance phase control capability) were calculated as the difference from baseline. Statistically and clinically significant improvements were observed after 10 treatment sessions in gait speed (0.25 ± 0.11 m/s) and FGA (4.55 ± 3.08 points). Statistically significant improvements were observed in TUG (2.36 ± 3.81 s) and FMLE (4.08 ± 1.82 points). A 10-session intervention combining tDCS and ML-based task-specific gait rehabilitation was feasible and produced clinically meaningful improvements in lower limb function in people with chronic gait deficits after stroke. Because only five users tested the new protocol, the results cannot be generalized to the whole population. As a contribution to the field, we developed and tested a protocol combining brain stimulation and ML-based stance phase training for individuals with chronic stance phase deficits after stroke. The protocol was feasible to administer; statistically and/or clinically significant improvements in gait function across an array of gait performance measures were observed with this relatively short treatment protocol.

Fifty-fifty - a comparison of two 50 watts high power short duration protocols using temperature- versus power- controlled radiofrequency ablation for atrial fibrillation

Abstract Funding Acknowledgements Type of funding sources: None. Background Radiofrequency-guided catheter ablation (RFCA) is an established treatment option for atrial fibrillation (AF). New approaches applying higher than usual energy levels for shorter periods (high power short duration, HPSD) to improve lesion quality have been reported. The novel DiamondTemp (DT) catheter allows for temperature-guided high power RFCA. Data on a direct comparison of the two emerging RFCA approaches are lacking. Purpose This observational single center study aimed to compare the efficacy, safety and characteristics of the novel DT catheter to an ablation index (AI) guided 50 W high power short duration (HPSD) ablation protocol using a conventional force-sensing equipped ablation catheter with surround-flow. Methods A total number of 101 patients undergoing RFA for AF were included. 33 patients treated with the DT catheter (50 W, 9 sec), were compared to 69 consecutive patients undergoing AI-guided AF ablation (AI anterior 550; AI posterior 400) with an open-irrigated catheter adherent to a 50 W HPSD protocol. Procedural data and recurrence rates were documented. Follow-up examinations were scheduled after 3, 6 and 12 months. Results Acute procedural success was achieved in all patients (n=101, 100%). DT-guided AF ablation was associated with a significantly longer mean procedure duration (98.8±30.1min vs. 78.2±25.6, p=0.002*). PVI using the DT ablation catheter required significantly more RF applications (75.4±30.8min vs. 61.3±14.1, p=0.019*). Total RF duration was significantly lower in the DT group (792.1±311.2sec vs. 1035.5±287.2sec, p<0.001*) as well as fluoroscopy time (4.6±2.1min vs.5.5±2.5min, p<0.006*) and dose (183.8±178.1yGym2 vs. 295.8±247.5yGym2, p<0.013*). Procedure related mayor complications occurred in 1 patient from the DT group (acute stroke; 3%) and in no patients from the HPSD cohort. Early recurrence was reported from 4 patients treated with the DT catheter (12%) compared to 8 patients undergoing HPSD RFA (12 %) (p=1.000). Conclusion Temperature- and power- controlled AF ablation using 50 W was safe and effective. AI-guided HPSD ablation resulted in significantly shorter procedure times with significantly fewer RF applications for PVI, whereas total RF duration and fluoroscopy times were significantly higher in this cohort. Further studies are needed to confirm this initial observation.

Guided Gradual Egg-Tolerance Induction in Hen's Egg Allergic Children Tolerating Baked Egg: A Prospective Randomized Trial.

BackgroundOver the last few years, studies have shown that the majority of egg allergic children tolerate baked egg (e.g., cake), and that consuming baked egg accelerates the resolution of egg allergy. However, few prospective studies demonstrate the step-wise reintroduction of egg at home after developing baked egg tolerance. Although this could have a positive impact on the children's quality of life and nutrition. Additionally, research supporting the theoretical concept that heating in the presence or absence of wheat causes reduced allergenicity of egg proteins is limited.ObjectiveTo investigate the clinically most favorable duration of gradual egg-tolerance induction in baked egg tolerant children at home, with regard to complete raw egg tolerance.MethodsBaked egg tolerant children above 12 months of age were randomly assigned to a short- or long arm protocol. In the short arm, egg-tolerance induction was studied over 18 months compared to 30 months in the long arm. Children were guided through this protocol involving the step-wise introduction of increasingly allergenic forms of egg starting with baked egg offered as cake, followed by hard-boiled egg, omelet/waffle/pancake, soft-boiled egg, and finally raw egg. We hereby designed this protocol based on the influence of thermal processing in the presence or absence of wheat on egg proteins, as investigated by ELISA, SDS-PAGE, and immunoblotting. At inclusion, children either passed an in-hospital cake challenge or had ovomucoid sIgE ≤1.2 kUA/L, which was considered safe for introduction at home.ResultsGel electrophoresis revealed that the ovalbumin band became weaker with heating, while the ovomucoid band remained stable. In accordance, the IgE-binding to ovalbumin decreased with extensive heating, as opposed to ovomucoid. However, heating in the presence of wheat led to a decreased IgE reactivity to ovomucoid. Of the 78 children in the intention-to-treat group, 39 were randomized to each arm. Fifty-eight children reached the raw egg tolerance endpoint, of which 80% were in the short arm and 69% in the long arm. Within the short arm, the median time to raw egg tolerance was 24 months (95% CI, 21–27 months) compared to 30 months (95% CI, 28–32 months) in the long arm (p = 0.005). No grade IV reactions or cases of eosinophilic esophagitis were observed. The short arm was considered to be non-inferior to the long arm.ConclusionOur gradual short arm protocol appears to be safe and allows clinicians to guide baked egg tolerant children toward raw egg tolerance at home. The allergenicity of the egg proteins was affected by heating temperature and duration, as well as the presence of wheat.

International proficiency trial for bovine viral diarrhea virus (BVDV) antibody detection: limitations of milk serology.

BackgroundControl programs were implemented in several countries against bovine viral diarrhea (BVD), one of the most significant cattle diseases worldwide. Most of the programs rely on serological diagnostics in any phase of the program. For the detection of antibodies against BVD virus (BVDV), neutralization tests as well as a variety of (commercially available) ELISAs are used. Here, test systems applied in various laboratories were evaluated in the context of an international interlaboratory proficiency trial. A panel of standardized samples comprising five sera and five milk samples was sent to veterinary diagnostic laboratories (n=51) and test kit manufacturers (n=3).ResultsThe ring trial sample panel was investigated by nine commercially available antibody ELISAs as well as by neutralization tests against diverse BVDV-1, BVDV-2 and/or border disease virus (BDV) strains. The negative serum and milk sample as well as a serum collected after BVDV-2 infection were mostly correctly tested regardless of the applied test system. A serum sample obtained from an animal immunized with an inactivated BVDV-1 vaccine tested positive by neutralization tests or by total antibody or Erns-based ELISAs, while all applied NS3-based ELISAs gave negative results. A further serum, containing antibodies against the ovine BDV, reacted positive in all applied BVDV ELISAs, a differentiation between anti-BDV and anti-BVDV antibodies was only enabled by parallel application of neutralization tests against BVDV and BDV isolates. For the BVDV antibody-positive milk samples (n=4), which mimicked prevalences of 20% (n=2) or 50% (n=2), considerable differences in the number of positive results were observed, which mainly depended on the ELISA kit and the sample incubation protocols used. These 4 milk samples tested negative in 43.6%, 50.9%, 3.6% and 56.4%, respectively, of all investigations. Overall, negative results occurred more often, when a short sample incubation protocol instead of an over-night protocol was applied.ConclusionsWhile the seronegative samples were correctly evaluated in most cases, there were considerable differences in the number of correct evaluations for the seropositive samples, most notably when pooled milk samples were tested. Hence, thorough validation and careful selection of ELISA tests are necessary, especially when applied during surveillance programs in BVD-free regions.

NtEdit+Sealer: Efficient Targeted Error Resolution and Automated Finishing of Long-Read Genome Assemblies.

High‐quality genome assemblies are crucial to many biological studies, and utilizing long sequencing reads can help achieve higher assembly contiguity. While long reads can resolve complex and repetitive regions of a genome, their relatively high associated error rates are still a major limitation. Long reads generally produce draft genome assemblies with lower base quality, which must be corrected with a genome polishing step. Hybrid genome polishing solutions can greatly improve the quality of long‐read genome assemblies by utilizing more accurate short reads to validate bases and correct errors. Currently available hybrid polishing methods rely on read alignments, and are therefore memory‐intensive and do not scale well to large genomes. Here we describe ntEdit+Sealer, an alignment‐free, k‐mer‐based genome finishing protocol that employs memory‐efficient Bloom filters. The protocol includes ntEdit for correcting base errors and small indels, and for marking potentially problematic regions, then Sealer for filling both assembly gaps and problematic regions flagged by ntEdit. ntEdit+Sealer produces highly accurate, error‐corrected genome assemblies, and is available as a Makefile pipeline from https://github.com/bcgsc/ntedit_sealer_protocol. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Automated long‐read genome finishing with short reads Support Protocol: Selecting optimal values for k‐mer lengths (k) and Bloom filter size (b)

Generation of Human iPSC-Derived Astrocytes with a mature star-shaped phenotype for CNS modeling

The generation of astrocytes from human induced pluripotent stem cells has been hampered by either prolonged differentiation—spanning over two months—or by shorter protocols that generate immature astrocytes, devoid of salient mature astrocytic traits pivotal for central nervous system (CNS) modeling. We directed stable hiPSC-derived neuroepithelial stem cells to human iPSC-derived Astrocytes (hiAstrocytes) with a high percentage of star-shaped cells by orchestrating an astrocytic-tuned culturing environment in 28 days. We employed RT-qPCR and ICC to validate the astrocytic commitment of the neuroepithelial stem cells. To evaluate the inflammatory phenotype, we challenged the hiAstrocytes with the pro-inflammatory cytokine IL-1β (interleukin 1 beta) and quantitatively assessed the secretion profile of astrocyte-associated cytokines and the expression of intercellular adhesion molecule 1 (ICAM-1). Finally, we quantitatively assessed the capacity of hiAstrocytes to synthesize and export the antioxidant glutathione. In under 28 days, the generated cells express canonical and mature astrocytic markers, denoted by the expression of GFAP, AQP4 and ALDH1L1. In addition, the notion of a mature phenotype is reinforced by the expression of both astrocytic glutamate transporters EAAT1 and EAAT2. Thus, hiAstrocytes have a mature phenotype that encompasses traits critical in CNS modeling, including glutathione synthesis and secretion, upregulation of ICAM-1 and a cytokine secretion profile on a par with human fetal astrocytes. This protocol generates a multifaceted astrocytic model suitable for in vitro CNS disease modeling and personalized medicine.Graphical abstract

A Concise Review on the Utilization of Abbreviated Protocol Breast MRI over Full Diagnostic Protocol in Breast Cancer Detection.

Breast MRI possesses high sensitivity for detecting breast cancer among the current clinical modalities and is an indispensable imaging practice. Breast MRI comprises diffusion-weighted imaging, ultrafast, and T2 weighted and T1 weighted CE (contrast-enhanced) imaging that may be utilized for improving the characterization of the lesions. This multimodal evaluation of breast lesions enables outstanding discrimination between the malignant and benign and malignant lesions. The expanding indications of breast MRI confirm the far superiority of MRI in preoperative staging, especially in the estimation of tumour size and identifying tumour foci in the contralateral and ipsilateral breast. Recent studies depicted that experts can meritoriously utilize this tool for improving breast cancer surgery despite their existence of no significant long term outcomes. For managing the, directly and indirectly, associated screening cost, abbreviated protocols are found to be more beneficial. Further, in some of the patients who were treated with neoadjuvant chemotherapy, breast MRI is utilized for documenting response. It is therefore essential to realise that oncological screening must be easily available, cost-effective, and time-consuming. Earlier detection of this short sequence protocol leads to prior and early breast cancer disease in high risky female populations like women with dense breasts, prehistoric evidence, etc. This proper utilization of AP reduces unnecessary mastectomies. Hence, this review focused on the explorative information for strongly suggesting the benefits of AP breast MRI compared to full diagnostic protocol MRI.

Considerations in evaluating equipment-free blood culture bottles: A short protocol for use in low-resource settings.

Use of equipment-free, "manual" blood cultures is still widespread in low-resource settings, as requirements for implementation of automated systems are often not met. Quality of manual blood culture bottles currently on the market, however, is usually unknown. An acceptable quality in terms of yield and speed of growth can be ensured by evaluating the bottles using simulated blood cultures. In these experiments, bottles from different systems are inoculated in parallel with blood and a known quantity of bacteria. Based on literature review and personal experiences, we propose a short and practical protocol for an efficient evaluation of manual blood culture bottles, aimed at research or reference laboratories in low-resource settings. Recommendations include: (1) practical equivalence of horse blood and human blood; (2) a diverse selection of 10 to 20 micro-organisms to be tested (both slow- and fast-growing reference organisms); (3) evaluation of both adult and pediatric bottle formulations and blood volumes; (4) a minimum sample size of 120 bottles per bottle type; (5) a formal assessment of usability. Different testing scenarios for increasing levels of reliability are provided, along with practical tools such as worksheets and surveys that can be used by laboratories wishing to evaluate manual blood culture bottles.

A follow-up study on congenital anomalies in 2208 offspring of three years old born after luteal-phase stimulation

Research questionWhat is the long-term safety in terms of congenital anomalies of a luteal-phase stimulation (LPS) protocol? DesignIn this cohort study, 664 couples and their children born after LPS and 1308 couples and their children born after a short agonist protocol were recruited. To investigate the long-term safety of LPS in terms of the prevalence of congenital anomalies, the physical growth and the health status of the offspring, the follow-up was divided into three steps: preparations before follow-up, first-stage follow-up including four telephone interviews, and second-stage follow-up when the children were aged 3 years. ResultsThe total prevalence of congenital anomalies did not differ between groups. The detailed classification showed a significantly lower percentage of musculoskeletal system congenital anomalies in singletons (P = 0.020) and an obviously higher percentage of digestive system congenital anomalies in multiple births (P = 0.028), both in the LPS group. In the evaluation of physical growth and health status, no significant differences were discovered between the two groups. ConclusionsThis study showed that offspring born after the LPS protocol did not exhibit an elevated rate of total congenital anomalies up to the age of 3. In addition, indicators regarding physical growth and health status were broadly similar between the two groups. These results have preliminarily confirmed the long-term safety of LPS. A subsequent long-term follow-up with a larger sample size should be carried out to generate more convincing evidence and more accurate conclusions.