Distribution of semen examination results 2020 – A follow up of data collated for the WHO semen analysis manual 2010

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.


| INTRODUC TI ON
Although the World Health Organisation (WHO) published the first laboratory manual for the examination of human semen and semencervical mucus interaction in 1980 (WHO 1980), the 5 th Edition 1 was the first to present distribution of semen parameter values that were based on significant supporting data. 2 These were defined from a population-based analysis of fertile men namely those with current or formerly pregnant partners with a known Time to Pregnancy (TTP) up to and including 12 months. 2 The 5 th centiles were presented as the lower reference values 1,2 and, as expected, these have been ubiquitously used in the literature and clinical practice.
It is 11 years since publication, and it is critical to determine whether they are still valid and/or whether they should be modified.
For example, there was a relatively limited range of subjects to formulate the WHO 2010 reference ranges -between 428 (for vitality) and 1941 (for semen volume). Moreover, a more global representation is required as there was no data from, for example, Africa or China.
To address this, we present substantial additional data obtained from published sources to potentially formulate more comprehensive and up to date lower reference values. These new data, combined with the previous information, comprises a database of semen analysis results of >3500 subjects, from twelve countries and five continents. This constitutes an updated and more comprehensive representation of the fertile man.

| Experimental design
This study presents two specific analyses. Analysis 1 presents a more recent reflection of semen parameters (post publication of WHO 2010), while Analysis 2 is a comprehensive presentation of values including data used for the WHO 2010 guidelines.
1. Analysis 1: Examination of published data following publication of the 5 th Edition of the WHO laboratory manual for the examination and processing of human semen. 1,2 We term this Analysis 1, 2010-2020 data.
2. Analysis 2: Examination of the data used to help formulate the 2010 2 reference values combined with the data from Analysis 1 (2010-2020 data). We term this Analysis 2, WHO 2020.

| Analysis 1: 2010-2020 data. Review and assessment of the literature
A literature review of papers that contained data on semen analysis was performed and the search strategy is described and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Flow Diagram 3 (Appendix S1 and S2). In addition, Scopus (Elsevier's abstract and citation database) was used to search for papers that cited Cooper et al. 2010. 2 The publication dates examined were 1 st January 2010 -30 th April 2020.
In this analysis we only assessed data from fertile men who have achieved a natural pregnancy with a known TTP ≤ 12 months and a sexual abstinence period of 2-7 days. Patients who were attending an infertility clinic and/or for fertility assessment were excluded.

| Data identification and processing
Analysis 1: 2010-2020 data Screening of the literature and analysis was performed by M. J. C. and C. L. R. B. Authors of the identified studies were contacted directly to obtain the raw semen analysis data related to their study (Table 1). All data examined was published in peer reviewed journals.
The data was collated within Microsoft Excel spreadsheets and statistical analysis was carried out using SPSSv25. The new data set presents a total of up to 1789 subjects, and incorporates data from three continents Africa, Asia and Europe.
The methodologies as described in the 5 th edition of the WHO 1 manual provided guidance on quality control and quality assurance.
When semen analysis was performed according to 5 th edition of WHO laboratory manual compliance to these procedures was assumed. PRISMA 3 Flow diagram is presented in Appendix S2.

| Analysis 2: WHO 2020
The dataset used to help formulate the WHO 2010 distribution of values was obtained from the editorial team of the sixth edition of the upcoming 'WHO laboratory manual for the examination and processing of human semen' . This data was examined and prepared to include only those semen analysis results with a confirmed TTP ≤ 12 months and a sexual abstinence range between 2 and 7 days. The data was then combined with 2010-2020 data (Analysis 1) to formulate WHO 2020 (Analysis 2). This combined data set presents a total of up to 3589 subjects, and incorporates data from five continents Africa, Oceania, Americas, Asia and Europe.
The data is freely available here https://doi.org/10.15132/ 10000163 and thus can be examined by investigators, added to and reanalysed, as and when appropriate.

| Result s
Since 2010, seven published studies have been identified as potentially suitable for detailed assessment. Data was kindly supplied from five published studies 4-8 (Table 1). Five studies documented using WHO standards for assessment (WHO 2010) however the morphology assessment by Aboutorabi et al. 5 was not compliant with WHO 2010 and therefore has been excluded. Vitality assessments were only available from the study by Evgeni et al. 8 Lotti et al. 4 , Tang et al. 7 and Zedan et al. 6 After accounting for an abstinence period of 2-7 days and a TTP of ≤12 months, up to 1789 subjects were analysed (Analysis 1: 2010-2020). Not all information was available for each subject. The key centiles are presented in Table 2.
The dataset used to help formulate the WHO 2010 values 1 was obtained from the editorial team of the sixth edition of the upcoming WHO laboratory manual. After accounting for an abstinence period of 2-7 days and confirming a TTP ≤ 12 months, up to 1800 subjects

| DISCUSS ION
This study presents substantial additional information to establish more comprehensive and globally applicable data for semen param-  The 5 th centile, is indicated above, and provides the lower reference values, of the fertile man.
as suggested in the WHO 2010 manual. Furthermore, there was no vitality information in the data provided by the editorial board.
Although the combined data (Analysis 2: WHO 2020, less than 20 million/ml. Suffice it to say we can suggest that, using these studies as benchmarks over the last 70 years, the 5 th centile for fertile men is realistically between 15-20 million/ml.
In summary, the distribution of semen assessment values now includes results from more than 3500 subjects, from twelve countries and five continents, providing a more global representation of the fertile man. Increasing the number of subjects provides robust information that is also more geographically representative. However, as emphasised, in the future, significantly larger studies encompassing data from regionally diverse populations will be needed to provide updated values.

D I SCL A I M ER
Some of the authors are present or former staff members of the World Health Organization. The authors alone are responsible for the views expressed in this publication and they do not necessarily represent the views, decisions or policies of the World Health Organization.

ACK N OWLED G EM ENTS
This study would not have been possible without the gracious and helpful support of the authors who performed the original data studies. They generously provided the raw data and were very helpful in interpretation. The authors are grateful for informal discussions with several colleagues including Niels Jørgensen, Phillip Matson and Christopher De Jonge.