Research Articles (Medical Bioscience)

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Editorial commentary on draft of world health organization sixth edition laboratory manual for the examination and processing of human semen
(Korean Society for Sexual Medicine and Andrology, 2021) Kandil, Hussein; Agarwal, Ashok; Henkel, Ralf
Semen examination is the cornerstone of the evaluation of male fertility potential. Despite its apparent simplicity, it is a complex series of assessments with highly variable results that are subject to interpretation. The semen analysis is used to gauge reproductive potential and guide the clinician in management of the infertile couple. Over the past 40 years, the World Health Organization (WHO) Infertility Task Force has attempted to standardize the methodology of semen examination so as to bring uniformity and relevance to the test.
The excessive use of antioxidant therapy: A possible cause of male infertility?
(Wiley, 2018) Henkel, Ralf; Sandhu, Inderpreet Singh; Agarwal, Ashok
Reactive oxygen species and oxidative stress are closely associated with various pathologies such as neurodegenerative diseases, ageing and male infertility. Hence, antioxidants such as vitamin C, vitamin E, N‐acetyl cysteine, L‐carnitine and folic acid are regularly used in various treatment regimens to protect cells from the damage induced by free radicals. However, given their over‐the‐counter availability at unnaturally high concentrations and also the fact that they are commonly added to various food products, patients may run a risk of consuming excessive dosages of these compounds, which may then be toxic. The few studies that have assessed antioxidant overuse and the associated adverse effects found that large doses of dietary antioxidant supplements have varying—if any—therapeutic effects even though free radicals clearly damage cells—a phenomenon that has been termed the “antioxidant paradox.” Furthermore, overuse of antioxidants such as vitamin C, vitamin E, N‐acetyl cysteine may lead to reductive stress, which is reported to be as dangerous to cells as oxidative stress and can be the cause of diseases such as cancer or cardiomyopathy. Therefore, we feel that there is a need for more elaborate research to establish the clear benefits and risks involved in antioxidant therapy for male infertility.
The impact of COVID-19 on the male reproductive tract and fertility: A systematic review
(Informa UK Limited, trading as Taylor & FrancisGroup., 2021) Sengupta, Pallav; Leisegang, Kristian; Agarwal, Ashok
Objective: The COVID-19 pandemic, caused by the acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), remains an ongoing public health challenge. Although males are affected slightly more than females, the impact of SARS-COV-2 on male reproductive system remains unclear. This systematic review aims to provide a concise update on the effects of COVID-19 on male reproductive health, including the presence of viral RNA in semen, and the impact on semen quality, testicular histology, testicular pain and male reproductive hormones. The global health is fronting an immediate as well as impending threat from the novel coronavirus (SARS- CoV-2) causing coronavirus disease (COVID-19), that inflicts more males than females. Evidence suggest that male reproductive system is susceptible to this viral infection. However, there are still several pertinent queries that remain to be fully explained regarding the mechanism in testicular SARS-CoV-2 dynamics and the exact mode of its actions. Thus, the present systematic review aims to provide a concise update on the effects of coronavirus disease 2019 (COVID-19) on male reproduction. Methods: A systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines searching the PubMed database. Eligibility for inclusion were original human studies evaluating the impact of COVID-19 on male repro- ductive health. Specific outcomes required for inclusion were at least one of the following: i) seminal detection of mRNA virus, or evaluation of ii) semen analysis, iii) testicular histology or ultrasonography, iv) testicular clinical symptoms and/or v) male reproductive hormones in COVID-19-positive patients. Results: Of 553 retrieved articles, 25 met the inclusion criteria. This included studies primarily investigating the presence of viral RNA in semen (n = 12), semen quality (n = 2), testicular histology (n = 5), testicular pain (n = 2) and male reproductive hormones (n= 4). Results show little evidence for the presence of viral RNA in semen, although COVID-19 seems to affect seminal parameters, induce orchitis, and cause hypogonadism. Mortality cases suggest severe histological disruption of testicular architecture, probably due to a systemic and local reproductive tract inflammatory response and oxidative stress-induced damage. Conclusions: Clinical evaluation of the male reproductive tract, seminal parameters and reproductive hormones is recommended in patients with current or a history of COVID-19,y treatment. Any long-term negative impact on male reproduction remains unexplored and an important future consideration.
An in-depth bibliometric analysis and current perspective on male infertility research
(Korean Society for Sexual Medicine and Andrology, 2019) Baskaran, Saradha; Agarwal, Ashok; Leisegang, Kristian; Peter, Natesan Pushparaj; Selvam, Manesh Kumar Panner; Henkel, Ralf
PURPOSE: Male infertility is emerging as a major, escalating global health problem that imposes the need to investigate research trends in male infertility. The purpose of this study is to analyze male infertility research trends in the past 20 years using the bibliometric database from Scopus. MATERIALS AND METHODS: In order to perform an in-depth bibliometric analysis, we propose a ‘Funnel Model’, which includes several layers representing different sub-areas of male infertility research. Adopting this Funnel Model, using Scopus, we retrieved relevant bibliometric data (articles per year, authors, affiliations, journals, and countries) for various areas of male infertility research and performed descriptive statistics. RESULTS: The bibliometric analysis showed an exponential increase in male infertility research in the last 20 years. USA dominated in research output, with Agarwal, A. as the most prolific researcher. Testicular cancer, obesity and metabolic syndrome, and azoospermia were found to dominate male infertility research, whereas erectile dysfunction and unexplained male infertility had lesser attention. Interestingly, prognostic/diagnostic and mechanistic studies have significantly increased in parallel over the last 20 years. Furthermore, our bibliometric analysis revealed fewer publications in proteomics, transcriptomics and metabolomics when compared to genomics. Also, an increasing trend in publication was seen in assisted reproductive technology (ART) research. CONCLUSIONS: An integrated and steep escalation in the field of omics and ART research appears to be a prerequisite for further development of future diagnostic and therapeutic strategies for male infertility.
Microtubular dysfunction and male infertility
(Korean Society for Sexual Medicine and Andrology, 2018) Gunes, Sezgin; Sengupta, Pallav; Henkel, Ralf; Alguraigari, Aabed; Sinigaglia, Mariana Marques; Kayal, Malik; Joumah, Ahmad; Agarwal, Ashok
Microtubules are the prime component of the cytoskeleton along with microfilaments. Being vital for organelle transport and cellular divisions during spermatogenesis and sperm motility process, microtubules ascertain functional capacity of sperm. Also, microtubule based structures such as axoneme and manchette are crucial for sperm head and tail formation. This review (a) presents a concise, yet detailed structural overview of the microtubules, (b) analyses the role of microtubule structures in various male reproductive functions, and (c) presents the association of microtubular dysfunctions with male infertility. Considering the immense importance of microtubule structures in the formation and maintenance of physiological functions of sperm cells, this review serves as a scientific trigger in stimulating further male infertility research in this direction.
Oxidative stress and assisted reproduction: A comprehensive review of its pathophysiological role and strategies for optimizing embryo culture environment
(MDPI, 2022) Agarwal, Ashok; Rosas, Israel Maldonado; Henkel, Ralf
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.
Oxidative stress: A comprehensive review of biochemical, molecular, and genetic aspects in the pathogenesis and management of varicocele
(Korean Society for Sexual Medicine and Andrology, 2021) Henkel, Ralf; Agarwal, Ashok; Sharma, Rakesh K; Gupta, Sajal
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.
Radiations and male fertility
(Springer Nature, 2018) Kesari, Kavindra Kumar; Agarwal, Ashok; Henkel, Ralf
During recent years, an increasing percentage of male infertility has to be attributed to an array of environmental, health and lifestyle factors. Male infertility is likely to be affected by the intense exposure to heat and extreme exposure to pesticides, radiations, radioactivity and other hazardous substances. We are surrounded by several types of ionizing and non-ionizing radiations and both have recognized causative effects on spermatogenesis. Since it is impossible to cover all types of radiation sources and their biological effects under a single title, this review is focusing on radiation deriving from cell phones, laptops, Wi-Fi and microwave ovens, as these are the most common sources of non-ionizing radiations, which may contribute to the cause of infertility by exploring the effect of exposure to radiofrequency radiations on the male fertility pattern. From currently available studies it is clear that radiofrequency electromagnetic fields (RF-EMF) have deleterious effects on sperm parameters (like sperm count, morphology, motility), affects the role of kinases in cellular metabolism and the endocrine system, and produces genotoxicity, genomic instability and oxidative stress. This is followed with protective measures for these radiations and future recommendations. The study concludes that the RF-EMF may induce oxidative stress with an increased level of reactive oxygen species, which may lead to infertility. This has been concluded based on available evidences from in vitro and in vivo studies suggesting that RF-EMF exposure negatively affects sperm quality.
Reactive oxygen species and male reproductive hormones
(BMC, 2018) Darbandi, Mahsa; Darbandi, Sara; Agarwal, Ashok; Sengupta, Pallav; Durairajanayagam, Damayanthi; Henkel, Ralf; Sadeghi, Mohammad Reza
Reports of the increasing incidence of male infertility paired with decreasing semen quality have triggered studies on the effects of lifestyle and environmental factors on the male reproductive potential. There are numerous exogenous and endogenous factors that are able to induce excessive production of reactive oxygen species (ROS) beyond that of cellular antioxidant capacity, thus causing oxidative stress. In turn, oxidative stress negatively affects male reproductive functions and may induce infertility either directly or indirectly by affecting the hypothalamus-pituitary-gonadal (HPG) axis and/or disrupting its crosstalk with other hormonal axes. This review discusses the important exogenous and endogenous factors leading to the generation of ROS in different parts of the male reproductive tract. It also highlights the negative impact of oxidative stress on the regulation and cross-talk between the reproductive hormones. It further describes the mechanism of ROS-induced derangement of male reproductive hormonal profiles that could ultimately lead to male infertility. An understanding of the disruptive effects of ROS on male reproductive hormones would encourage further investigations directed towards the prevention of ROS-mediated hormonal imbalances, which in turn could help in the management of male infertility.
Reactive oxygen species and male reproductive hormones
(BMC, 2018) Darbandi, Mahsa; Darbandi, Sara; Agarwal, Ashok; Sengupta, Pallav; Durairajanayagam, Damayanthi; Henkel, Ralf; Sadeghi, Mohammad Reza
Reports of the increasing incidence of male infertility paired with decreasing semen quality have triggered studies on the effects of lifestyle and environmental factors on the male reproductive potential. There are numerous exogenous and endogenous factors that are able to induce excessive production of reactive oxygen species (ROS) beyond that of cellular antioxidant capacity, thus causing oxidative stress. In turn, oxidative stress negatively affects male reproductive functions and may induce infertility either directly or indirectly by affecting the hypothalamus-pituitary-gonadal (HPG) axis and/or disrupting its crosstalk with other hormonal axes. This review discusses the important exogenous and endogenous factors leading to the generation of ROS in different parts of the male reproductive tract. It also highlights the negative impact of oxidative stress on the regulation and cross-talk between the reproductive hormones. It further describes the mechanism of ROS-induced derangement of male reproductive hormonal profiles that could ultimately lead to male infertility. An understanding of the disruptive effects of ROS on male reproductive hormones would encourage further investigations directed towards the prevention of ROS-mediated hormonal imbalances, which in turn could help in the management of male infertility.
Scientific landscape of oxidative stress in male reproductive research: A scientometric study
(Elsevier, 2020) Henkel, Ralf; Agarwal, Ashok; Baskaran, Saradha
Unraveling the role of reactive oxygen species and associated oxidative stress (OS) in male reproduction is one of the key areas of male reproductive research. This article illustrates the scientific landscape of OS in male reproductive research over the past several decades (1941–2018) using a scientometric approach. Scientometric data (articles per year, authors, affiliations, journals, and countries) on OS related to male reproduction were retrieved from the Scopus database and analyzed for each decade. Our analysis revealed an increasing trend in OS-based male reproductive research from 1941 to 2018 with a steep raise in publications and research collaborations starting from the period 1991–2000 (R2 = 0.81). Semen abnormalities and varicocele were the major areas investigated in relation to OS with the highest positive trend in publications from the time interval 1981–1990 to 2011–2018. Analysis of publications based on OS assessment techniques revealed chemiluminescence (n = 180) and evaluation of antioxidants (n = 300) as the most widely used direct and indirect tests, respectively.
Seminal oxidation–reduction potential levels are not influenced by the presence of leucocytospermia
(Wiley-Blackwell, 2020) Henkel, Ralf; Arafa, Mohamed Mostafa; Agarwal, Ashok
Oxidative stress (OS) is characterised by an excessive amount of reactive oxygen species (ROS) which negatively affect sperm functions. In this study, the influence of leucocytes on seminal oxidation–reduction potential (ORP) and sperm DNA fragmentation (SDF) was investigated in 1,068 men. Seminal leucocyte concentration did not correlate with SDF, unadjusted ORP, ORP normalised for sperm concentration (sORP), ORP normalised for total motile sperm concentration (motORP) or total motile sperm count (TMSC-ORP). Although receiver operator characteristic (ROC) curve analyses show that leucocytospermia does not predict high sORP values (>1.34 mV/106 spermatozoa/ml), the motORP (AUC: 0.666) and TMSC-ORP (AUC: 0.683) predict the rate of leucocytospermia significantly (p =.0195 and p =.0085 respectively). Moreover, SDF can significantly predict leucocytospermia (AUC: 0.679; p =.011) and vice versa (AUC: 0.657, p =.0298). Our data confirm the association between OS and SDF. In conclusion, motORP and TMSC-ORP may be better predictive factors of leucocytospermia, probably because sperm motility, included in motORP and TMSC-ORP calculation, is the first seminal parameter to be affected by OS. Although all these parameters are indicative of OS, ORP values, SDF and leucocytospermia should be considered independently for the evaluation of redox seminal status, as they probe distinct seminal features.
Sperm morphology assessment in the era of intracytoplasmic sperm injection: Reliable results require focus on standardization, quality control, and training
(Korean Society for Sexual Medicine and Andrology, 2021) Agarwal, Ashok; Sharma, Rakesh; Henkel, Ralf
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.
Utility of Antioxidants in the Treatment of Male Infertility: Clinical Guidelines Based on a Systematic Review and Analysis of Evidence
(The world journal of men's health, 2021) Agarwal, Ashok; Leisegang, Kristian
It is widely accepted that oxidative stress plays an important role in the pathophysiology of male infertility and that antioxidants could have a significant role in the treatment of male infertility. The main objectives of this study are: 1) to systematically review the current evidence for the utility of antioxidants in the treatment of male infertility; and 2) propose evidence-based clinical guidelines for the use of antioxidants in the treatment of male infertility. A systematic review of the available clinical evidence was performed, with articles published on Scopus being manually screened. Data extracted included the type of antioxidant used, the clinical conditions under investigation, the evaluation of semen parameters and reproductive outcomes. The adherence to the Cambridge Quality Checklist, Cochrane Risk of Bias for randomized controlled trials (RCTs), CONSORT guidelines and JADAD score were analyzed for each included study. Further, we provided a Strength Weakness Opportunity Threat (SWOT) analysis to analyze the current and future value of antioxidants in male infertility. Of the 1,978 articles identified, 97 articles were included in the study. Of these, 52 (53.6%) were uncontrolled (open label), 12 (12.4%) unblinded RCTs, and 33 (34.0%) blinded RCTs, whereas 44 (45.4%) articles tested individual antioxidants, 31 (32.0%) a combination of several products in variable dosages, and 22 (22.6%) registered antioxidant products. Based on the published evidence, we 1) critically examined the necessity of addi- tional double-blind, randomized, placebo-controlled trials, and 2) proposed updated evidence-based clinical guidelines for anti- oxidant therapy in male infertility. The current systematic review on antioxidants and male infertility clearly shows that antioxidant supplementation improves semen parameters. In addition, it provides the indications for antioxidant treatment in specific clinical conditions, including varicocele, unexplained and idiopathic male infertility, as well as in cases of altered semen quality.
The validity and reliability of computer-aided semen analyzers in performing semen analysis: a systematic review
(Translational Andrology and Urology., 2021) Leisegang, Kristian; Agarwal, Ashok; Renata, Finelli
Background: Computer-aided sperm analyzers (CASA) are currently used worldwide for semen analysis. However, there are doubts about their reliability to fully substitute the human operator. Therefore, this study aimed to systematically review the current literature comparing results from semen evaluation by both CASA-based and manual approaches. Methods: A systematic screening of the literature was performed based on the PRISMA guidelines and by searching on PubMed, Scopus, and Embase databases. Results: A total of 14 studies were included. Our results showed a high degree of correlation for sperm concentration and motility when analysis was performed either manually or by using a CASA system. However, CASA results showed increased variability in low (<15 million/mL) and high (>60 million/ mL) concentration specimens, while sperm motility assessment was inaccurate in samples with higher concentration or in the presence of non-sperm cells and debris. Morphology results showed the highest level of difference, due to the high amount of heterogeneity seen between the shapes of the spermatozoa either in one sample or across multiple samples from the same subject. Conclusions: Overall, our study suggests CASA systems as a valid alternative for the evaluation of semen parameters in clinical practice, especially for sperm concentration and motility. However, further technological improvements are required before these devices can one day completely replace the human operator. Artificial intelligence-based CASA devices promise to offer higher efficiency of the analysis and improve the reliability of results.

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