Abstract
For the reproducibility and sustainability of scientific research, FAIRness (Findable, Accessible, Interoperable and Re-usable), with respect to the release of raw data obtained by ...researchers, is one of the most important principles underpinning the future of open science. In genomics and transcriptomics, the sharing of raw data from next-generation sequencers is made possible through public repositories. In addition, in proteomics, the deposition of raw data from mass spectrometry (MS) experiments into repositories is becoming standardized. However, a standard repository for such MS data had not yet been established in glycomics. With the increasing number of glycomics MS data, therefore, we have developed GlycoPOST (https://glycopost.glycosmos.org/), a repository for raw MS data generated from glycomics experiments. In just the first year since the release of GlycoPOST, 73 projects have already been registered by researchers around the world, and the number of registered projects is continuously growing, making a significant contribution to the future FAIRness of the glycomics field. GlycoPOST is a free resource to the community and accepts (and will continue to accept in the future) raw data regardless of vendor-specific formats.
•Telomere length has been investigated as a cellular aging biomarker for environmental exposure and life style factors.•Inconsistencies using the qPCR telomere length method highlight the need for a ...careful methodological analysis of the whole process.•We summarize each critical step in qPCR telomere length assay and provide guidance and recommendations based on current knowledge.•We suggest that the telomere research community collaborate on the development and implementation of standardized protocols.
Research in the last decade has explored the length of telomeres, the protective ends of eukaryotic chromosomes, as a biomarker for the cumulative effects of environmental exposures and life experiences as well as a risk factor for major diseases. With a growing interest in telomere biology across biomedical, epidemiological and public health research, it is critical to ensure that the measurement of telomere length is performed with high precision and accuracy. Of the several major methods utilized to determine telomere length, quantitative PCR (qPCR) remains the most cost-effective and suitable method for large-scale epidemiological and population studies. However, inconsistencies in recent reports utilizing the qPCR method highlight the need for a careful methodological analysis of each step of this process. In this review, we summarize each critical step in qPCR telomere length assay, including sample type selection, sample collection, storage, processing issues and assay procedures. We provide guidance and recommendations for each step based on current knowledge. It is clear that a collaborative and rigorous effort is needed to characterize and resolve existing issues related to sample storage, both before and after DNA extraction, as well as the impact of different extraction protocols, reagents and post extraction processing across all tissue types (e.g. blood, saliva, buccal swabs, etc.) to provide the needed data upon which best practices for TL analyses can be agreed upon. Additionally, we suggest that the whole telomere research community be invited to collaborate on the development and implementation of standardized protocols for the assay itself as well as for reporting in scientific journals. The existing evidence provides substantial support for the continuation of telomere research across a range of different exposures and health outcomes. However, as with any technological or methodologic advance in science, reproducibility, reliability and rigor need to be established to ensure the highest quality research.
Reducing the number of spermatozoa per artificial insemination (AI) dose and managing semen in ways to ensure greater quality at the same time represents current challenges with sperm processing in ...pig AI centers. Based on a multi-year comparative analysis of process steps in different pig AI centers, and complementary experimental studies under standardized laboratory conditions, current process standards for the preservation of boar semen have been updated and new ones developed. Currently, these standards represent an integral part of the quality assurance of 29 European pig AI centers in ten different organizations in Germany, Switzerland and Austria. Improvement of hygiene management and guidelines for prudent use of antibiotics have become key issues. Furthermore, new quality control tools have been implemented in the processing and transport of boar semen: e.g. refractometry as an easy-to-use tool to estimate extender osmolarity and ‘mobile sensing’ apps for continuous monitoring of various environmental parameters. Moreover, based on a series of experiments under laboratory and field conditions, guidelines for optimizing the dilution process, and time and temperature management during boar semen processing, have been developed and implemented. Similarly, recommendations for the handling of semen doses during storage have been renewed. Over the years, the efficiency of the quality assurance system has been reflected by a decrease of bacterial contamination and a concomitant increase in the quality of semen doses. In conclusion, science-based quality assurance is an effective way to improve the production performance in pig AI centers, resulting in high quality and economically-priced semen for pig producers. Increasing knowledge of sperm physiology together with computational and technical innovations will continue to develop and modify quality assurance concepts in the future.
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe international shortage of the nasopharyngeal swabs that are required for collection of optimal ...specimens, creating a critical bottleneck blocking clinical laboratories' ability to perform high-sensitivity virological testing for SARS-CoV-2. To address this crisis, we designed and executed an innovative, cooperative, rapid-response translational-research program that brought together health care workers, manufacturers, and scientists to emergently develop and clinically validate new swabs for immediate mass production by 3D printing. We performed a multistep preclinical evaluation of 160 swab designs and 48 materials from 24 companies, laboratories, and individuals, and we shared results and other feedback via a public data repository (http://github.com/rarnaout/Covidswab/). We validated four prototypes through an institutional review board (IRB)-approved clinical trial that involved 276 outpatient volunteers who presented to our hospital's drive-through testing center with symptoms suspicious for COVID-19. Each participant was swabbed with a reference swab (the control) and a prototype, and SARS-CoV-2 reverse transcriptase PCR (RT-PCR) results were compared. All prototypes displayed excellent concordance with the control (κ = 0.85 to 0.89). Cycle threshold (
) values were not significantly different between each prototype and the control, supporting the new swabs' noninferiority (Mann-Whitney U MWU test,
> 0.05). Study staff preferred one of the prototypes over the others and preferred the control swab overall. The total time elapsed between identification of the problem and validation of the first prototype was 22 days. Contact information for ordering can be found at http://printedswabs.org Our experience holds lessons for the rapid development, validation, and deployment of new technology for this pandemic and beyond.
Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell ...culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise.
Background
Uterine leiomyomas, also referred to as myomas or fibroids, are benign tumours arising from the smooth muscle cells of the myometrium. They are the most common pelvic tumour in women. The ...estimated rate of leiomyosarcoma, found during surgery for presumed benign leiomyomas, is about 0.51 per 1000 procedures, or approximately 1 in 2000.
Treatment options for symptomatic uterine leiomyomas include medical, surgical, and radiologically‐guided interventions. Laparoscopic myomectomy is the gold standard surgical approach for women who want offspring, or otherwise wish to retain their uterus. A limitation of laparoscopy is the inability to remove large specimens from the abdominal cavity through the laparoscope. To overcome this challenge, the morcellation approach was developed, during which larger specimens are broken into smaller pieces in order to remove them from the abdominal cavity via the port site. However, intracorporeal power morcellation may lead to scattering of benign tissues, with the risk of spreading leiomyoma or endometriosis. In cases of unsuspected malignancy, power morcellation can cause unintentional dissemination of malignant cells, and lead to a poorer prognosis by upstaging the occult cancer.
A strategy to optimise women's safety is to morcellate the specimens inside a bag. In‐bag morcellation may avoid the dissemination of tissue fragments.
Objectives
To evaluate the effectiveness and safety of protected in‐bag extracorporeal manual morcellation during laparoscopic myomectomy compared to intra‐abdominal uncontained power morcellation.
Search methods
On 1 July 2019, we searched; the Cochrane Gynaecology and Fertility Group Specialized Register of Controlled Trials, CENTRAL, MEDLINE, Embase, PsycINFO, CINAHL, LILACS, PubMed, Google Scholar, and two trials registers.
We reviewed the reference lists of all retrieved full‐text articles, and contacted experts in the field for additional and ongoing trials.
Selection criteria
We included all randomised controlled trials comparing in‐bag extracorporeal manual morcellation versus intracorporeal uncontained power morcellation during laparoscopic myomectomy in premenopausal women.
Data collection and analysis
We followed standard Cochrane methods.
Two review authors independently reviewed the eligibility of trials, extracted data, and evaluated the risk of bias. Data were checked for accuracy.
The summary measures were reported as risk ratios (RR) or mean differences (MD) with 95% confidence interval (CI).
The outcomes of interest were a composite of intraoperative and postoperative complications, operative times, ease of morcellation, length of hospital stay, postoperative pain, conversion to laparotomy, and postoperative diagnosis of leiomyosarcoma. Results for the five main outcomes follow.
Main results
We included two trials, enrolling 176 premenopausal women with fibroids, who underwent laparoscopic myomectomy.
The experimental group received in‐bag manual morcellation, during which each enucleated myoma was placed into a specimen retrieval bag, and manually morcellated with scalpel or scissors. In the control group, intracorporeal uncontained power morcellation was used to reduce the size of the myomas.
No intraoperative complications, including accidental morcellation of the liver, conversion to laparotomy, endoscopic bag disruption, bowel injury, bleeding, accidental injury to any viscus or vessel, were reported in either group in either trial.
We found very low‐quality evidence of inconclusive results for total operative time (MD 9.93 minutes, 95% CI ‐1.35 to 21.20; 2 studies, 176 participants; I² = 35%), and ease of morcellation (MD ‐0.73 points, 95% CI ‐1.64 to 0.18; 1 study, 104 participants). The morcellation operative time was a little longer for the in‐bag manual morcellation group, however the quality of the evidence was very low (MD 2.59 minutes, 95% CI 0.45 to 4.72; 2 studies, 176 participants; I² = 0%). There were no postoperative diagnoses of leiomyosarcoma made in either group in either trial.
We are very uncertain of any of these results. We downgraded the quality of the evidence due to indirectness and imprecision, because of limited sites in high‐income settings and countries, small sample sizes, wide confidence intervals, and few events.
Authors' conclusions
There are limited data on the effectiveness and safety of in‐bag morcellation at the time of laparoscopic myomectomy compared to uncontained power morcellation. We were unable to determine the effects of in‐bag morcellation on intraoperative complications as no events were reported in either group. We are uncertain if in‐bag morcellation improves total operative time or ease of morcellation compared to control. Regarding morcellation operative time, the quality of the evidence was also very low and we cannot be certain of the effect of in‐bag morcellation compared to uncontained morcellation. No cases of postoperative diagnosis of leiomyosarcoma occurred in either group. We found only two trials comparing in‐bag extracorporeal manual morcellation to intracorporeal uncontained power morcellation at the time of laparoscopic myomectomy. Both trials had morcellation operative time as primary outcome and were not powered for uncommon outcomes such as intraoperative complications, and postoperative diagnosis of leiomyosarcoma.
Large, well‐planned and executed trials are needed.
•Sample-to-sample amount variation could be larger than analytical variation.•Sample normalization is a critical step in quantitative metabolomics.•Sample normalization should be incorporated into a ...metabolomic profiling workflow.•There is no unified method but a number of methods have been reported.•The performance of sample normalization methods needs to be carefully considered to select a proper method.
To reveal metabolomic changes caused by a biological event in quantitative metabolomics, it is critical to use an analytical tool that can perform accurate and precise quantification to examine the true concentration differences of individual metabolites found in different samples. A number of steps are involved in metabolomic analysis including pre-analytical work (e.g., sample collection and storage), analytical work (e.g., sample analysis) and data analysis (e.g., feature extraction and quantification). Each one of them can influence the quantitative results significantly and thus should be performed with great care. Among them, the total sample amount or concentration of metabolites can be significantly different from one sample to another. Thus, it is critical to reduce or eliminate the effect of total sample amount variation on quantification of individual metabolites. In this review, we describe the importance of sample normalization in the analytical workflow with a focus on mass spectrometry (MS)-based platforms, discuss a number of methods recently reported in the literature and comment on their applicability in real world metabolomics applications. Sample normalization has been sometimes ignored in metabolomics, partially due to the lack of a convenient means of performing sample normalization. We show that several methods are now available and sample normalization should be performed in quantitative metabolomics where the analyzed samples have significant variations in total sample amounts.
Antibiotic agents such as gentamicin represent essential components of semen extenders in order to reduce bacterial contamination. But antibiotic resistance increases and AI centers start utilizing ...antibiotic agents which are more potent. Therefore, a shift to preventing bacterial contamination has to take place. In this study, we could demonstrate that hygiene is a tool capable of reducing bacterial load. In order to analyze 1434 extended semen samples and nine specially established hygienic critical control points (HCCPs, n = 828), 92 quality control audits have been carried out in a time period from 2012 until 2019 in 28 European AI centers. The results show the process of introducing a basic hygienic standard in audit 1 (2012/2013) and 2 (2014/2015) and the resulting achievements by means of improved hygienic conditions in audit 3 (2016/2017) and 4 (2018/2019). Within the scope of audit 1, 19% of the semen samples were contaminated with bacteria (cutoff ≥100 colony-forming units/mL). Audit 2 showed a bacterial load of 13.6% whereas during audit 3 and 4 very low bacterial contamination rates were recorded (4.5 and 5.5%, respectively). In the same manner, analysis of hygiene at different CCPs during semen production showed a decrease in all average HCCP-scores (score 1–6) comparing audit 4 to 1. By regression analysis we could show a significant audit-dependent association of the bacterial contamination in semen samples and hygiene of HCCPs. Furthermore, analysis of the odds ratio (OR) reveals that the bacterial contamination of certain HCCPs poses an increased risk of receiving bacterially contaminated semen samples (filling machine: OR = 3.02, P = 0.06; extender: OR = 8.97, P < 0.001; inner face of dilution tank lids: OR = 3.14, P = 0.09). Around 60% of the variance of the bacterial contamination in semen samples could be explained by hygienic conditions at different control points and their interaction with audit period and AI center. Antimicrobial agents are essential to protect human and animal health but excessive or inappropriate use can lead to the emergence of resistant bacteria. As shown in our study, hygiene management can significantly reduce bacterial contamination and is therefore capable of preventing antibiotic resistance.
•Regularly evaluated AI centers show low over-all contamination rates.•Hygiene at defined HCCPs is closely related with bacterial load in AI doses.•Hygiene is a potent tool being capable of reducing bacterial load in AI doses.
Artificial reproduction involves collection and handling of gametes in a way that secures their quality and maximizes the fertilization outcome. In addition to initial sperm quality, numerous steps ...can affect the final result of fertilization, from the sperm collection process until gamete mixing (or co-incubation) when the spermatozoon enters or fuses with the oocyte. In this review, we summarize the whole process of sperm handling, from collection until fertilization for fish, penaeid shrimp, bivalve mollusks and marine mammals. To obtain sperm from captive animals, techniques vary widely across taxa, and include stripping by abdominal massage or testis surgical removal in fish, spermatophore collection in penaeid shrimps, gonadal scarification or temperature shock in bivalve mollusks, and voluntary collection via positive reinforcement in mammals. In most cases, special care is needed to avoid contamination by mucus, seawater, urine, or feces that can either activate sperm motility and/or decrease its quality. We also review techniques and extender solutions used for refrigerated storage of sperm across the aforementioned taxa. Finally, we give an overview of the different protocols for in vivo and in vitro fertilization including activation of sperm motility and methods for gamete co-incubation. The present study provides valuable information regarding breeder management either for animal production or species conservation.
•In vitro and in vivo fertilization involve sperm collection and handling until gamete co-incubation.•Practical guidelines for sperm handling in fish, penaeid shrimp, bivalve mollusks and marine mammals are presented.•Sperm refrigeration techniques are revised for the different taxa.•Protocols for gamete co-incubation are discussed across the different taxa.
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