Small airways in asthma: From inflammation and pathophysiology to treatment response.

Small airways are characterized as those with an inner diameter less than 2 mm and constitute a major site of pathology and inflammation in asthma disease. It is estimated that small airways dysfunction may occur before the emergence of noticeable symptoms, spirometric abnormalities and imaging findings, thus characterizing them as "the quiet or silent zone" of the lungs. Despite their importance, measuring and quantifying small airways dysfunction presents a considerable challenge due to their inaccessibility in usual functional measurements, primarily due to their size and peripheral localization. Several pulmonary function tests have been proposed for the assessment of the small airways, including impulse oscillometry, nitrogen washout, body plethysmography, as well as imaging methods. Nevertheless, none of these methods has been established as the definitive "gold standard," thus, a combination of them should be used for an effective assessment of the small airways. Widely used asthma treatments seem to also affect several parameters of the small airways. Emerging biologic treatments show promising results in reducing small airways inflammation and remodelling, providing evidence for potential alterations in the disease's progression and outcomes. These novel therapies have implications not only in the clinical aspects of asthma but also in its inflammatory and functional aspects.

Oocytes evaluation and in-vitro fertilization/intra cytoplasmic sperm injection outcomes.

The identification of markers capable of evaluating oocyte quality, its maturation, function, and embryo progression and implantation potential has frequently initiated research interest. However, to date, univocal criteria of oocyte competence do not exist. A major cause of low oocyte quality is evidently advanced maternal age. However, other factors may influence oocyte competence. Among these are obesity, lifestyle factors, genetic and systematic pathologies, ovarian stimulation protocols, laboratory procedures, culture, and environmental conditions. The morphological and maturational evaluation of oocytes is probably the most widely used. Several morphological features, both cytoplasmic (cytoplasmic pattern and hue, presence of vacuoles, refractile bodies, granulation, and smooth endoplasmic reticulum clusters) and extra-cytoplasmic (perivitelline space, zona pellucida thickness, oocyte shape, and polar bodies), have been proposed to distinguish oocytes with the best reproductive potential among a cohort. No single abnormality seems to be sufficiently predictive of the developmental capacity of the oocyte. Some abnormalities such as cumulus cells dysmorphisms, central granulation, vacuoles, and smooth endoplasmic reticulum clusters, however, seem to be associated with poor developmental potential of the embryo, although oocyte dysmorphisms are very common and the data in the literature is limited and provide conflicting views. Other criteria involving gene expression of cumulus cells as well as the metabolomic analysis of spent culture media have been explored. Also, sophisticated technologies such as polar bodies biopsy, meiotic spindle visualization, mitochondrial activity, oxygen consumption, and measurement of glucose-6-phosphate dehydrogenase activity have been proposed. Many of these approaches, however, remain largely research-based and have not found widespread application in clinical service. Due to the lack of consistent data for the assessment of oocyte quality and competence, probably oocyte morphology and oocyte maturity remain important indicators to determine oocyte quality. The aim of this review was to provide spherical attributes and evidence on recent and present research on the topic by analyzing the current methods for evaluation of the oocyte quality, and the impact of oocyte quality on reproductive outcomes. Additionally, current limitations of oocyte quality evaluation are highlighted and insights on future research are provided to optimize the selection techniques of oocytes to improve ART outcomes.

Renoprotective Effect of Agalsidase Alfa: A Long-Term Follow-Up of Patients with Fabry Disease.

Fabry disease is a rare lysosomal storage disorder caused by mutations in the GLA gene, which, without treatment, can cause significant renal dysfunction. We evaluated the effects of enzyme replacement therapy with agalsidase alfa on renal decline in patients with Fabry disease using data from the Fabry Outcome Survey (FOS) registry. Male patients with Fabry disease aged >16 years at agalsidase alfa start were stratified by low (≤0.5 g/24 h) or high (>0.5 g/24 h) baseline proteinuria and by 'classic' or 'non-classic' phenotype. Overall, 193 male patients with low (n = 135) or high (n = 58) baseline proteinuria were evaluated. Compared with patients with low baseline proteinuria, those with high baseline proteinuria had a lower mean ± standard deviation baseline eGFR (89.1 ± 26.2 vs. 106.6 ± 21.8 mL/min/1.73 m2) and faster mean ± standard error eGFR decline (−3.62 ± 0.42 vs. −1.61 ± 0.28 mL/min/1.73 m2 per year; p < 0.0001). Patients with classic Fabry disease had similar rates of eGFR decline irrespective of baseline proteinuria; only one patient with non-classic Fabry disease had high baseline proteinuria, preventing meaningful comparisons between groups. In this analysis, baseline proteinuria significantly impacted the rate of eGFR decline in the overall population, suggesting that early treatment with good proteinuria control may be associated with renoprotective effects.

Twenty years of the Fabry Outcome Survey (FOS): insights, achievements, and lessons learned from a global patient registry.

BACKGROUND: Patient registries provide long-term, real-world evidence that aids the understanding of the natural history and progression of disease, and the effects of treatment on large patient populations with rare diseases. The year 2021 marks the 20th anniversary of the Fabry Outcome Survey (FOS), an international, multicenter, observational registry (NCT03289065). The primary aims of FOS are to broaden the understanding of Fabry disease (FD), an X-linked lysosomal storage disorder, and to improve the clinical management of affected patients. Here, we review the history of FOS and the analyses and publications disseminated from the registry, and we discuss the contributions FOS studies have made in understanding FD. RESULTS: FOS was initiated in April 2001 and, as of January 2021, 4484 patients with a confirmed diagnosis and patient informed consent have been enrolled from 144 centers across 26 countries. Data from FOS have been published in nearly 60 manuscripts on a wide variety of topics relevant to FD. Analyses of FOS data have investigated the long-term effectiveness and safety of enzyme replacement therapy (ERT) with agalsidase alfa and its effects on morbidity and mortality, as well as the benefits of prompt and early treatment with agalsidase alfa on the progression of cardiomyopathy and the decline in renal function associated with FD. Based on analyses of FOS data, ERT with agalsidase alfa has also been shown to improve additional signs and symptoms of FD experienced by patients. FOS data analyses have provided a better understanding of the natural history of FD and the specific populations of women, children, and the elderly, and have provided practical tools for the study of FD. FOS has also provided methodology and criteria for assessing disease severity which contributed to the continuous development of medical practice in FD and has largely improved our understanding of the challenges and needs of long-term data collection in rare diseases, aiding in future rare disease real-world evidence studies. CONCLUSION: FOS over the last 20 years has substantially increased the scientific knowledge around improved patient management of FD and continues to expand our understanding of this rare disease.

Oxidative Stress and Assisted Reproduction: A Comprehensive Review of Its Pathophysiological Role and Strategies for Optimizing Embryo Culture Environment.

Oxidative stress (OS) due to an imbalance between reactive oxygen species (ROS) and antioxidants has been established as an important factor that can negatively affect the outcomes of assisted reproductive techniques (ARTs). Excess ROS exert their pathological effects through damage to cellular lipids, organelles, and DNA, alteration of enzymatic function, and apoptosis. ROS can be produced intracellularly, from immature sperm, oocytes, and embryos. Additionally, several external factors may induce high ROS production in the ART setup, including atmospheric oxygen, CO2 incubators, consumables, visible light, temperature, humidity, volatile organic compounds, and culture media additives. Pathological amounts of ROS can also be generated during the cryopreservation-thawing process of gametes or embryos. Generally, these factors can act at any stage during ART, from gamete preparation to embryo development, till the blastocyst stage. In this review, we discuss the in vitro conditions and environmental factors responsible for the induction of OS in an ART setting. In addition, we describe the effects of OS on gametes and embryos. Furthermore, we highlight strategies to ameliorate the impact of OS during the whole human embryo culture period, from gametes to blastocyst stage.

An expert commentary on essential equipment, supplies and culture media in the assisted reproductive technology laboratory.

The assisted reproductive technology (ART) laboratory is a complex system designed to sustain the fertilization, survival, and culture of the preimplantation embryo to the blastocyst stage. ART outcomes depend on numerous factors, among which are the equipment, supplies and culture media used. The number and type of incubators also may affect ART results. While large incubators may be more suitable for media equilibration, bench-top incubators may provide better embryo culture conditions in separate or smaller chambers and may be coupled with time-lapse systems that allow continuous embryo monitoring. Microscopes are essential for observation, assessment, and micromanipulation. Workstations provide a controlled environment for gamete and embryo handling and their quantity should be adjusted according to the number of ART cycles treated in order to provide a steady and efficient workflow. Continuous maintenance, quality control and monitoring of equipment are essential and quality control devices such as the thermometer, and pH-meter are necessary to maintain optimal culture conditions. Tracking, appropriate delivery and storage conditions, and quality control of all consumables are recommended so that adequate quantity and quality are available for use. Embryo culture media have evolved: preimplantation embryos are cultured either by sequential media or single-step media that can be used for interrupted or uninterrupted culture. There is currently no sufficient evidence that any individual commercially-available culture system is better than others in terms of embryo viability. In this review, we aim to analyze the various parameters that should be taken into account when choosing the essential equipment, consumables and culture media systems that will create optimal culture conditions and provide the most effective patient treatment.

Optimizing embryological aspects of oocyte retrieval, oocyte denudation, and embryo loading for transfer.

Oocyte retrieval, oocyte denudation, and embryo transfer are crucial processes during assisted reproduction technology (ART). Air quality in the ART laboratory, temperature, pH of the media used and the time interval between oocyte retrieval and insemination are all critical factors. Anesthesia is required for oocyte retrieval, however, evidence regarding the potential impact of different methods (general anesthesia, conscious sedation, and local anesthesia) on the clinical outcomes is unclear. The optimal timing of oocyte denudation following retrieval has not been established. Regarding the mechanical denudation process, there is a lack of evidence to demonstrate the safest minimum inner diameter of denuding pipettes used to complete the removal of granulosa cells surrounding the oocytes. During embryo transfer, many clinics worldwide flush the catheter before embryo loading, in an attempt to potentially rinse off any toxic agents; however, there is insufficient evidence to show that flushing the embryo transfer catheter before loading increases the success of ART outcome. Considering the serious gaps in knowledge in ART practice, the aim of this review is to provide an updated overview of the current knowledge regarding the various steps and techniques involved in oocyte retrieval, oocyte denudation, and embryo loading for transfer.

Clinical aspects of oocyte retrieval and embryo transfer: tips and tricks for the novice and the expert.

Oocyte retrieval (ovum pick-up) and embryo transfer (ET) are essential steps in in-vitro fertilization and intracytoplasmic sperm injection and over the years, the two procedures were developed in order to improve the clinical outcome. Many suggestions were proposed and applied before, during and after oocyte retrieval such as timing of HCG trigger, pre-operative pelvic scan, vaginal cleansing, type of anesthesia, type and gauge of aspiration needles, aspiration pressure, follicle flushing, and the need for prophylactic antibiotics. Similarly, many steps were suggested and implemented before, during and after ET including patient's position, type of anesthesia/analgesia, dummy (mock) ET, ultrasound-guidance, HCG injection in the uterine cavity, use of relaxing agents, full bladder, removal of the cervical mucus, flushing the cervix with culture medium, type of ET catheter, embryo loading techniques, site of embryo deposition, the use of adherence compounds, as well as bed rest after ET. Complications were also reported with oocyte retrieval and ET. The aim of this review is to evaluate the current practice of these two procedures in the light of available evidence.

Oocyte quality and embryo selection strategies: a review for the embryologists, by the embryologists.

With the advance of assisted reproduction techniques, and the trend towards blastocyst culture and single embryo transfer, gamete and embryo assessment have gained greater importance in ART treatment. Embryo quality depends mainly on gamete quality and culture conditions. Oocyte maturity identification is necessary in order to plan fertilization timing. Mature oocytes at the metaphase II stage show a higher fertilization rate compared to immature oocytes. Morphology assessment is a critical yet challenging task that may serve as a good prognostic tool for future development and implantation potential if done effectively. Various grading systems have been suggested to assess embryos at pronuclear, cleavage, and blastocyst stages. By identifying the embryo with the highest implantation potential, it is possible to reduce the number of embryos transferred without compromising the chances of a successful pregnancy. Apart from the conventional morphology assessment, there are several invasive or non-invasive methods for embryo selection such as preimplantation genetic testing, morphokinetics, proteomics, metabolomics, oxygen consumption, and measurement of oxidative stress in culture medium. Morphokinetics is a method based on time-lapse technology and continuous monitoring of embryos. In this review, we aimed to describe and compare the most effective and widely used methods for gamete and embryo assessment as well as embryo selection.

Taking a closer look at the key performance indicators in an assisted reproductive technology laboratory: a guide for reproductive professionals.

Over the years, laboratories performing assisted reproductive technologies have been tasked with a growing number of procedures of increased complexity. New technologies, including hardware and software innovations, are constantly evolving, and evaluated as potential tools to improve laboratory and clinical outcomes. The assisted reproductive technology (ART) laboratory plays a crucial role in fertility treatments and, therefore, it is often under intense scrutiny with regards to performance and success rates. As the reproductive medicine field strives to deliver improved clinical outcomes to patients, in-vitro fertilization (IVF) clinics - including the ART laboratories - are required to monitor their performance and seek improvement in the many different aspects related to patient care. Key performance indicators (KPIs) and benchmarking are important tools to support performance monitoring and quality improvement processes. The concept and potential benefits of KPI utilization is generally accepted. However, its adoption poses some challenges that may discourage ART practices from pursuing it as part of their quality management systems (QMS). Properly selecting and using KPIs will allow laboratories to successfully manage their performance and set up realistic target goals to consistently deliver high rates. Existing literature can guide reproductive medicine professionals to embark on their journey to successfully select, implement, and manage KPI and benchmarking as part of their ART programs. This article discusses the concept and essentials of KPI and benchmarking applied to the ART laboratory, as well as potential challenges and how to overcome them.

Sperm Vitality and Necrozoospermia: Diagnosis, Management, and Results of a Global Survey of Clinical Practice.

Sperm vitality testing is a basic semen examination that has been described in the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen from its primary edition, 40 years ago. Several methods can be used to test sperm vitality, such as the eosin-nigrosin (E-N) stain or the hypoosmotic swelling (HOS) test. In the 6th (2021) edition of the WHO Laboratory Manual, sperm vitality assessment is mainly recommended if the total motility is less than 40%. Hence, a motile spermatozoon is considered alive, however, in certain conditions an immotile spermatozoon can also be alive. Therefore, the differentiation between asthenozoospermia (pathological decrease in sperm motility) and necrozoospermia (pathological decrease in sperm vitality) is important in directing further investigation and management of infertile patients. The causes leading to necrozoospermia are diverse and can either be local or general, testicular or extra-testicular. The andrological management of necrozoospermia depends on its etiology. However, there is no standardized treatment available presently and practice varies among clinicians. In this study, we report the results of a global survey to understand current practices regarding the physician order of sperm vitality tests as well as the management practices for necrozoospermia. Laboratory and clinical scenarios are presented to guide the reader in the management of necrozoospermia with the overall objective of establishing a benchmark ranging from the diagnosis of necrozoospermia by sperm vitality testing to its clinical management.

A Comprehensive Guide to Sperm Recovery in Infertile Men with Retrograde Ejaculation.

Retrograde ejaculation (RE) is a condition defined as the backward flow of the semen during ejaculation, and when present can result in male infertility. RE may be partial or complete, resulting in either low seminal volume or complete absence of the ejaculate (dry ejaculate). RE can result from anatomic, neurological or pharmacological conditions. The treatment approaches outlined are determined by the cause. Alkalinizing urinary pH with oral medications or by adding sperm wash media into the bladder prior to ejaculation may preserve the viability of the sperm. This article provides a step-by-step guide to diagnose RE and the optimal techniques to retrieve sperm.

Relevance of Leukocytospermia and Semen Culture and Its True Place in Diagnosing and Treating Male Infertility.

The current WHO 2010 manual for human semen analysis defines leukocytospermia as the presence of peroxidase-positive leukocytes at a concentration >1×106/mL of semen. Granular leukocytes when activated are capable of generating high levels of reactive oxygen species in semen resulting in oxidative stress. Oxidative stress has been correlated with poor sperm quality, increased level of sperm DNA fragmentation and low fertility potential. The presence of leukocytes and pathogens in the semen may be a sign of infection and/or localized inflammatory response in the male genital tract and the accessory glands. Common uro-pathogens including Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Mycoplasma hominis, and Escherichia coli can cause epididymitis, epididymo-orchitis, or prostatitis. The relationship between leukocytospermia and infection is unclear. Therefore, we describe the pathogens responsible for male genital tract infections and their association with leukocytospermia. The review also examines the diagnostic tests available to identify seminal leukocytes. The role of leukocytospermia in male infertility and its management is also discussed.

Sperm Morphology Assessment in the Era of Intracytoplasmic Sperm Injection: Reliable Results Require Focus on Standardization, Quality Control, and Training.

Semen analysis is the first, and frequently, the only step in the evaluation of male fertility. Although the laboratory procedures are conducted according to the World Health Organization (WHO) guidelines, semen analysis and especially sperm morphology assessment is very difficult to standardize and obtain reproducible results. This is mainly due to the highly subjective nature of their evaluation. ICSI is the choice of treatment when sperm morphology is severely abnormal (teratozoospermic). Hence, the standardization of laboratory protocols for sperm morphology evaluation represents a fundamental step to ensure reliable, accurate and consistent laboratory results that avoid misdiagnoses and inadequate treatment of the infertile patient. This article aims to promote standardized laboratory procedures for an accurate evaluation of sperm morphology, including the establishment of quality control and quality assurance policies. Additionally, the clinical importance of sperm morphology results in assisted reproductive outcomes is discussed, along with the clinical management of teratozoospermic patients.

Impact of the COVID-19 pandemic on the standard of care for patients with lysosomal storage diseases: A survey of healthcare professionals in the Fabry, Gaucher, and Hunter Outcome Survey registries.

The impact of the COVID-19 pandemic on the standards of care of patients with lysosomal storage diseases and the needs of their healthcare providers were explored using a 12-question survey. Overall, 80/91 respondents (88%) indicated that the pandemic had negatively affected standards of care. With increased reliance on telemedicine, the respondents highlighted the need for a personalized approach to care, direct and frequent communication with patients, and greater involvement of patients and caregivers.

A Web-Based Global Educational Model for Training in Semen Analysis during the COVID-19 Pandemic.

PURPOSE: In response to the COVID-19 pandemic, the American Center for Reproductive Medicine (ACRM) transitioned its annual training in assisted reproductive technology (ART) from a hands-on, laboratory-based training course to a fully online training endorsed by the American College of Embryology. Here we describe our experience and assess the quality of an online training format based on participant outcomes for the first three modules of a planned series of online ART training. MATERIALS AND METHODS: These modules included manual semen analysis, sperm morphology and ancillary semen tests (testing for leukocytospermia, sperm vitality, and anti-sperm antibody screening). The virtual format consisted of lecture presentations featuring laboratory protocols with corresponding video demonstrations of routine techniques and best practices. Practical scenarios, troubleshooting, and clinical interpretation of laboratory results were also discussed. At the end of each module, an optional multiple choice question test was held as a prerequisite to obtain certification on the topics presented. Course quality was assessed using participant responses collected via online surveys. RESULTS: The digital delivery methods used were found to have largely or completely met the participants' expectations for all questions (>85%). The majority (>87%) of the participants either strongly agreed or agreed that the course content was well-structured with appropriate depth, and that their overall expectations of the course had been met. CONCLUSIONS: This training format appears to be a realistic teaching option to freely share highly specialized expertise and technical knowledge with participants from anywhere in the world with varying levels of competency or experience.