Background It is now 11 years since publication of the WHO 2010 guidelines for semen assessment values, and it is critical to determine whether they are still valid and/or whether they should be modified. Objectives To utilise data published since 2010 and combine these with data used in the 2010 assessment to provide an updated and more comprehensive representation of the fertile man. This may be utilised to present an updated distribution of values for use by WHO in 2021. Materials and Methods Two specific analyses were performed namely, (1) Analysis 1: Examination of published data following publication of WHO 2010 [termed 2010–2020 data]. (2) Analysis 2: Examination of the data used to help formulate the 2010 distribution of values combined with the data from Analysis (1) [termed WHO 2020]. Results In total, data from more than 3500 subjects, from twelve countries and five continents were analysed. The 5th centile values for concentration, motility and morphology are: 16 × 106/ml, 30% progressive motility [42% total motility] and 4% normal forms. Discussion This study presents substantial additional information to establish more comprehensive and globally applicable lower reference values for semen parameters for fertile men although they do not represent distinct limits between fertile and subfertile men. There are still data missing from many countries and, some geographical regions are not represented. Moreover, the number of subjects although significant is still relatively low (<4000). Conclusion These distributions of values now include semen analysis providing a more global representation of the fertile man. Increasing the number of subjects provides robust information that is also more geographically representative.

STUDY QUESTION Do publications that involve the interpretation of the results of a basic semen analysis, published in Human Reproduction and Fertility & Sterility between 2011 and 2020, give sufficient evidence in their methodology to demonstrate that they followed the technical methods recommended in the fifth edition of the World Health Organization (WHO) laboratory manual, entitled WHO Laboratory Manual for the Examination and Processing of Human Semen (WHO5)? SUMMARY ANSWER Evidence of methodological agreement of studies with the WHO5 recommendations was low, despite 70% of papers stating that they followed WHO5 recommendations. WHAT IS KNOWN ALREADY A basic semen analysis is currently an integral part of infertility investigations of the male, but method standardization in laboratories remains an issue. The different editions of the WHO manual for the basic semen analysis (WHO1–6) have attempted to address this by providing increasingly rigorous methodological protocols to reduce experimental error. However, to what extent these methods are followed by studies that involve the interpretation of the results of basic semen analysis remains unknown. STUDY DESIGN, SIZE, DURATION A survey of the technical methods used to perform a basic semen analysis was conducted on studies published in two leading reproduction journals (Human Reproduction and Fertility & Sterility) between 2011 and 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS The literature search was performed on the electronic databases PUBMED and MEDLINE Ovid between January 2021 and March 2021. The MeSH terms included in the search were ‘sperm concentration’ OR ‘sperm motility’ OR ‘sperm morphology’ OR ‘sperm vitality’ OR ‘male fertility’ AND ‘human spermatozoa’ NOT ‘animals’. A total of 122 studies were available for analysis. MAIN RESULTS AND THE ROLE OF CHANCE In total, 70% of the studies cited WHO5 in their methods section. Of the remaining studies, 10% cited the fourth edition of the WHO laboratory manual (WHO4), 7% cited both WHO4 and WHO5, 1% cited the third edition of the WHO laboratory manual (WHO3), and 12% did not cite the WHO at all. Overall methodological agreement with WHO5 recommendations was poor, with the main reason for this lack of agreement being that the research studies did not disclose specific details of the technical methods and equipment used. LIMITATIONS, REASONS FOR CAUTION In the case of studies that did not disclose any specific technical methods that they used, we did not attempt to contact these authors and so were unable to confirm the agreement between their technical methods and WHO5 recommendations. WIDER IMPLICATIONS OF THE FINDINGS Our findings suggest there is an urgent need to develop strategies to address standardization in reporting the results of a semen analysis for publication. This is particularly timely given the recent publication of WHO6 and ISO standard 23162 for the basic examination of human semen. STUDY FUNDING/COMPETING INTEREST(S) There was no funding for this project. C.L.R.B., as an employee of the University of Dundee, serves on the Scientific Advisory board of ExSeed Health (from October 2021, financial compensation to the University of Dundee) and is a scientific consultant for Exscientia (from September 2021, financial compensation to the University of Dundee). C.L.R.B. has previously received a fee from Cooper Surgical for lectures on scientific research methods outside the submitted work (2020) and Ferring for a lecture on male reproductive health (2021). C.L.R.B. is Editor for RBMO. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTION Does AZD5904, a myeloperoxidase inhibitor (MPOi), have any effect on human sperm function in vitro? SUMMARY ANSWER AZD5904 improves sperm function in an in vitro model of oxidative stress (OS) and potentially offers a novel treatment approach for male infertility. WHAT IS KNOWN ALREADY Male infertility is an underlying or contributory cause in half of all couples experiencing difficulties conceiving, yet there is currently no effective treatment or cure. OS is a common pathology in a significant proportion of infertile men. It can negatively affect sperm motility and the ability to fertilize a mature oocyte, as well as DNA integrity, and therefore represents an attractive target for therapeutic intervention. STUDY DESIGN, SIZE, DURATION This study included population-based samples from men (23–50 years) attending Ninewells Assisted Conception Unit, Dundee for diagnostic semen analysis, July 2017–September 2018. Semen samples (n = 47) from 45 patients were used. PARTICIPANTS/MATERIALS, SETTING, METHODS Neutrophils activated using zymosan were incubated with prepared human spermatozoa for 2 h (T2) and 24 h (T24) to create an in vitro model of OS. Parallel samples were co-incubated with AZD5904, an MPOi, to examine its effects. Sperm motility was assessed by computer-assisted sperm analysis at T2 and T24. Functional motility was assessed by sperm penetration assay. Statistical analysis was performed using GraphPad Prism. MAIN RESULTS AND THE ROLE OF CHANCE There was no significant difference in total or progressive sperm motility between any treatment and control groups at T2 or T24. Nonetheless, significant positive effects on sperm function were observed with AZD5904, with 16/45 (35.6%) samples (with both normal and abnormal baseline semen analysis characteristics) displaying a ≥20% increase in sperm penetrated through viscous media (P &lt; 0.003). LIMITATIONS, REASONS FOR CAUTION This was an in vitro study. WIDER IMPLICATIONS OF THE FINDINGS Treatment with AZD5904 resulted in significant increased sperm penetration in one of three samples treated, which is likely to represent improvement in sperm function required for fertilization. We are now planning a clinical trial to validate these results and hope that this could represent a new treatment for male infertility. STUDY FUNDING/COMPETING INTEREST(S) AZD5904 was shared through the AstraZeneca Open Innovation program. The study was funded by AstraZeneca and sponsored by the University of Dundee. Additional funding was provided by Chief Scientist Office/NHS Research Scotland (S.J.M.d.S.). A.W. and H.J.S. are both full time employees of AstraZeneca. A.W. and H.J.S. are inventors on a patent filed by AstraZeneca titled MPOi for use in medicine which includes MPOi for use in the treatment of male infertility (WO 2019/016074 Al). S.J.M.d.S. is Associate Editor of Human Reproduction and Editorial Board member of Reproduction & Fertility. C.L.R.B. is Editor of RBMO and has received lecturing fees from Merck and Ferring and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B. was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012–2016). C.L.R.B. has a patent WO2013054111 A1 issued. The other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.