Standards in semen examination:publishing reproducible and reliable data based on high-quality methodology

Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.Peer reviewe

Evaluation of sperm nuclear DNA integrity in infertile men: correlation with conventional seminal parameters

Background: Conventional semen analysis has been traditionally considered as the first step in the evaluation of male infertility. Due to its limitations in predicting a man’s fertility potential, sperm DNA fragmentation was recently introduced as a novel marker of sperm quality. Aim of the study: To investigate the possible correlations between conventional seminal parameters and DNA fragmentation in specific subgroups of Greek men, selected in relation to the levels of their basic seminal profile and their fertility history.Materials and Methods: A total of 1,439 subject data were evaluated in different subgroups: normozoospermic (n=184) and non-normozoospermic (n=485), according to the WHO 2010 reference ranges and based on fertility status: fertile donors and men with children naturally conceived within one year of unprotected intercourse (n=78), subfertile men, having achieved pregnancies either naturally or by ART, not resulting in live births (n=153) and infertile men, failing to produce either pregnancies or children (n=539). Semen volume, sperm concentration, total count, rapid and total progressive motility and morphology was evaluated following the WHO 2010 methods. DNA fragmentation was assessed by the SCD assay. Correlations were sought between the attributes in all subgroups.Results: The WHO 2010 reference limits for the conventional semen analysis are adequately descriptive of the seminal normality in the Greek population. An inverse correlation was established between DNA fragmentation and all the conventional seminal parameters except semen volume, in men with seminal profiles below the reference ranges and also in men of diminished fertility potential. Statistical significance was observed for rapid and total progressive motility in non-normospermic subjects, as well as sperm concentration, total count, progressive motility (rapid and total) and normal morphology in subfertile and infertile subjects. In our group of fertile men, no statistically significant correlations were remarked between conventional semen parameters and the levels of DNA fragmentation. Unscreened normozoospemic men exhibited lower levels of DNA fragmentation than their non-normozoospermic counterparts, even though the values were not always found below 30%.Conclusions: DNA fragmentation testing and traditional semen analysis should be considered as complementary tools in the investigation of male infertility, with associations varying according to the studied population subgroups. A comprehensive evaluation of male infertility should include the assessment of both attributes of semen quality.Εισαγωγή: Η συμβατική ανάλυση σπέρματος έχει παραδοσιακά θεωρηθεί ως το πρώτο βήμα στη διερεύνηση της ανδρικής υπογονιμότητας. Λόγω των περιορισμών που παρουσιάζει στη δυνατότητα πρόγνωσης του γονιμοποιητικού δυναμικού ενός άνδρα, εισήχθη πρόσφατα στην εργαστηριακή ανδρολογία η αξιολόγηση της κατάτμησης του σπερματικού DNA ως ένας νέος δείκτης ποιότητας του ανδρικού σπέρματος.Σκοπός της μελέτης: Η αναζήτηση πιθανών συσχετίσεων μεταξύ των συμβατικών σπερματολογικών παραμέτρων και της κατάτμησης DNA, σε διακριτές υπο-ομάδες Ελλήνων ανδρών, επιλεγμένων βάσει των επιπέδων του σπερματικού τους προφίλ και του γονιμοποιητικού τους ιστορικού.Υλικά και Μέθοδοι: Δεδομένα από ένα σύνολο 1.439 περιστατικών αξιολογήθηκαν σε διαφορετικές υπο-ομάδες ανδρών, που περιελάμβαναν: νορμοζωοσπερμικούς (ν=184) και μη νορμοζωοσπερμικούς (ν=485), σύμφωνα με τα όρια αναφοράς του Π.Ο.Υ. 2010, καθώς επίσης και με γνώμονα το ιστορικό γονιμότητας: γόνιμους δότες και άνδρες που είχαν αποκτήσει παιδιά με φυσιολογική σύλληψη εντός ενός έτους ελεύθερων επαφών (ν=78), υπογόνιμους άνδρες, οι οποίοι είχαν επιτύχει εγκυμοσύνη είτε φυσιολογικά είτε μέσω ΙΥΑ, η οποία δεν κατέληξε σε γέννηση (ν=153) και μη γόνιμους άνδρες, στο ιστορικό των οποίων δεν είχε αναφερθεί ούτε εγκυμοσύνη ούτε γέννηση (ν=539). Ο όγκος σπέρματος, η συγκέντρωση των σπερματοζωαρίων, ο ολικός αριθμός, η ταχεία και ολική προωθητική κινητικότητα και η μορφολογία αξιολογήθηκαν σύμφωνα με τις προτεινόμενες από το εγχειρίδιο του Π.Ο.Υ. 2010 μεθόδους. Η κατάτμηση του DNA των σπερματοζωαρίων αξιολογήθηκε με τη μέθοδο Sperm Chromatin Dispersion – SCD. Αναζητήθηκαν συσχετίσεις μεταξύ των παραμέτρων σε όλες τις πληθυσμιακές υπο-ομάδες.Αποτελέσματα: Τα προτεινόμενα από τον Π.Ο.Υ. 2010 όρια αναφοράς για τις συμβατικές σπερματολογικές παραμέτρους περιγράφουν με επάρκεια τη φυσιολογική σπερματική ποιότητα στον Ελληνικό πληθυσμό. Αρνητική συσχέτιση καταγράφηκε μεταξύ της κατάτμησης του σπερματικού DNA και όλων των βασικών παραμέτρων εκτός του σπερματικού όγκου, σε άνδρες με σπερματολογικά προφίλ κάτω των ορίων αναφοράς και σε άνδρες με μειωμένο γονιμοποιητικό δυναμικό. Στατιστική σημαντικότητα παρατηρήθηκε για την ταχεία και ολική προωθητική κινητικότητα σε μη νορμοζωοσπερμικούς άνδρες και για τη συγκέντρωση, τον ολικό αριθμό, την προωθητική κινητικότητα (ταχεία και ολική) και τη φυσιολογική μορφολογία σε υπογόνιμους και μη γόνιμους άνδρες. Στη ομάδα ελέγχου των γόνιμων ανδρών, δεν διαπιστώθηκαν στατιστικά σημαντικές συσχετίσεις μεταξύ των συμβατικών παραμέτρων και του βαθμού της κατάτμησης DNA. Οι νορμοζωοσπερμικοί, με μη καθορισμένο ιστορικό γονιμότητας, άνδρες εμφάνισαν χαμηλότερα επίπεδα κατάτμησης DNA σε σύγκριση με τους αντίστοιχους μη νορμοζωοσπερμικούς, παρ’ ότι οι τιμές τους δεν ανιχνεύονταν πάντα κάτω του 30%.Συμπεράσματα: Η εξέταση του βαθμού κατάτμησης του σπερματικού DNA και η παραδοσιακή ανάλυση σπέρματος δύνανται να θεωρηθούν συμπληρωματικά μεθοδολογικά εργαλεία κατά τη διερεύνηση της ανδρικής υπογονιμότητας, με συσχετίσεις που ποικίλλουν ανάλογα με τις υπό μελέτη πληθυσμιακές υπο-ομάδες. Μία εμπεριστατωμένη αξιολόγηση της ανδρικής υπογονιμότητας οφείλει να συμπεριλαμβάνει και τις δύο αυτές πτυχές της σπερματικής ποιότητας

Human Sperm DNA Fragmentation and its Correlation with Conventional Semen Parameters

Abstract Background: The initial step in the diagnostic investigation of male infertility has been traditionally based on the conventional seminal profile. However, there are significant limitations regarding its ability to determine the underlying mechanisms that cause the disorder. Sperm DNA fragmentation has emerged as a potential causative factor of reproductive failure and its assessment has been suggested as a useful adjunct to the laboratory methodology of male infertility evaluation, especially before the application of assisted reproduction technology (ART). Methods: A review of recent bibliography was carried out in PubMed by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation. Results: A comprehensive diagnostic approach of male infertility should be based on a combination of diagnostic attributes, derived from the conventional semen analysis, as well as the investigation of genomic integrity testing. Conclusion: Due to its strong correlation with several aspects of ART procedures and further consequences for the offspring, sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies focusing on specific subgroups of infertile men who may benefit from an efficient therapeutic management based on the optimization of sperm DNA integrity are needed

Sperm DNA fragmentation test: usefulness in assessing male fertility and assisted reproductive technology outcomes

International audienceMale infertility is attributed to multiple factors including high levels of sperm DNA fragmentation (SDF). Conventional semen analysis continues to be the gold standard for diagnosis of male factor infertility around the world. However, the limitations of basic semen analysis have prompted the search for complementary assessments of sperm function and integrity. Sperm DNA fragmentation assays (direct or indirect) are emerging as important diagnostic tools in male infertility workups, and have been advocated for use in infertile couples for a variety of reasons. While a controlled degree of DNA nicking is required for appropriate DNA compaction, excessive fragmentation of sperm DNA is linked to impaired male fertility potential, decreased fertilization, poor embryo quality, recurrent pregnancy loss, and failure of assisted reproductive technology procedures. However, there is an ongoing debate regarding whether or not to employ SDF as a routine test for male infertility. This review compiles up-to-date information regarding the pathophysiology of SDF, the currently available SDF tests, and the role of SDF tests in natural and assisted conception conditions

Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men

Background: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. ----- Methods: The study was conducted with 166 participants, 20–55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. ----- Results: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%. ----- Conclusion: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics

Redox Balance in Male Infertility: Excellence through Moderation—“Μέτρον ἄριστον”

Male infertility, a relatively common and multifactorial medical condition, affects approximately 15% of couples globally. Based on WHO estimates, a staggering 190 million people struggle with this health condition, and male factor is the sole or contributing factor in roughly 20–50% of these cases. Nowadays, urologists are confronted with a wide spectrum of conditions ranging from the typical infertile male to more complex cases of either unexplained or idiopathic male infertility, requiring a specific patient-tailored diagnostic approach and management. Strikingly enough, no identifiable cause in routine workup can be found in 30% to 50% of infertile males. The medical term male oxidative stress infertility (MOSI) was recently coined to describe infertile men with abnormal sperm parameters and oxidative stress (OS), including those previously classified as having idiopathic infertility. OS is a critical component of male infertility, entailing an imbalance between reactive oxygen species (ROS) and antioxidants. ROS abundance has been implicated in sperm abnormalities, while the exact impact on fertilization and pregnancy has long been a subject of considerable debate. In an attempt to counteract the deleterious effects of OS, urologists resorted to antioxidant supplementation. Mounting evidence indicates that indiscriminate consumption of antioxidants has led in some cases to sperm cell damage through a reductive-stress-induced state. The “antioxidant paradox”, one of the biggest andrological challenges, remains a lurking danger that needs to be carefully avoided and thoroughly investigated. For that reason, oxidation-reduction potential (ORP) emerged as a viable ancillary tool to basic semen analysis, measuring the overall balance between oxidants and antioxidants (reductants). A novel biomarker, the Male infertility Oxidative System (MiOXSYS®), is a paradigm shift towards that goal, offering a quantification of OS via a quick, reliable, and reproducible measurement of the ORP. Moderation or “Μέτρον” according to the ancient Greeks is the key to successfully safeguarding redox balance, with MiOXSYS® earnestly claiming its position as a guarantor of homeostasis in the intracellular redox milieu. In the present paper, we aim to offer a narrative summary of evidence relevant to redox regulation in male reproduction, analyze the impact of OS and reductive stress on sperm function, and shed light on the “antioxidant paradox” phenomenon. Finally, we examine the most up-to-date scientific literature regarding ORP and its measurement by the recently developed MiOXSYS® assay

Fertile man semen parameters 2020 – an update to the data collated for the WHO semen analysis manual 2010

This dataset contains the semen parameters of a fertile man, who have achieved a pregnancy in ≤ 12 months of trying to conceive, with an abstinence period of 2-7 days, at time of semen analysis. The parameters of a semen analysis included are; Semen Volume (ml), Sperm concentration(10x6 per ml), Total sperm number (10x6 per ejaculate), Total motility (PR + NP, %), Progressive motility (PR, %), Non-progressive motility (NP, %), Immotile spermatozoa (IM, %), Vitality (%) and Normal forms (%). The dataset has been derived from the subjects used to formulate the normal semen parameters of a fertile man, stated within The WHO laboratory manual for the examination and processing of human semen, Fifth Edition and from a systematic review of published papers from 01/01/2010 to 30/04/2020, which included semen parameters of fertile men, which were conducted according to standards set within The WHO laboratory manual for the examination and processing of human semen, Fifth Edition

Antisperm antibody testing: A comprehensive review of its role in the management of immunological male infertility and results of a global survey of clinical practices

Antisperm antibodies (ASA), as a cause of male infertility, have been detected in infertile males as early as 1954. Multiple causes of ASA production have been identified, and they are due to an abnormal exposure of mature germ cells to the immune system. ASA testing (with mixed anti-globulin reaction, and immunobead binding test) was described in the WHO manual 5th edition and is most recently listed among the extended semen tests in the WHO manual 6th edition. The relationship between ASA and infertility is somewhat complex. The presence of sperm agglutination, while insufficient to diagnose immunological infertility, may indicate the presence of ASA. However, ASA can also be present in the absence of any sperm agglutination. The andrological management of ASA depends on the etiology and individual practices of clinicians. In this article, we provide a comprehensive review of the causes of ASA production, its role in immunological male infertility, clinical indications of ASA testing, and the available therapeutic options. We also provide the details of laboratory procedures for assessment of ASA together with important measures for quality control. Additionally, laboratory and clinical scenarios are presented to guide the reader in the management of ASA and immunological male infertility. Furthermore, we report the results of a recent worldwide survey, conducted to gather information about clinical practices in the management of immunological male infertility

Consensus and diversity in the management of varicocele for male infertility: Results of a global practice survey and comparison with guidelines and recommendations

Purpose: Varicocele is a common problem among infertile men. Varicocele repair (VR) is frequently performed to improve semen parameters and the chances of pregnancy. However, there is a lack of consensus about the diagnosis, indications for VR and its outcomes. The aim of this study was to explore global practice patterns on the management of varicocele in the context of male infertility. Materials and Methods: Sixty practicing urologists/andrologists from 23 countries contributed 382 multiple-choice-questions pertaining to varicocele management. These were condensed into an online questionnaire that was forwarded to clinicians involved in male infertility management through direct invitation. The results were analyzed for disagreement and agreement in practice patterns and, compared with the latest guidelines of international professional societies (American Urological Association [AUA], American Society for Reproductive Medicine [ASRM], and European Association of Urology [EAU]), and with evidence emerging from recent systematic reviews and meta-analyses. Additionally, an expert opinion on each topic was provided based on the consensus of 16 experts in the field. Results: The questionnaire was answered by 574 clinicians from 59 countries. The majority of respondents were urologists/uro-andrologists. A wide diversity of opinion was seen in every aspect of varicocele diagnosis, indications for repair, choice of technique, management of sub-clinical varicocele and the role of VR in azoospermia. A significant proportion of the responses were at odds with the recommendations of AUA, ASRM, and EAU. A large number of clinical situations were identified where no guidelines are available. Conclusions: This study is the largest global survey performed to date on the clinical management of varicocele for male infertility. It demonstrates: 1) a wide disagreement in the approach to varicocele management, 2) large gaps in the clinical practice guidelines from professional societies, and 3) the need for further studies on several aspects of varicocele management in infertile men