Evolution of the WHO “Semen” processing manual from the first (1980) to the sixth edition (2021)

Abstract

As stated clearly in all editions of the WHO Laboratory Manual for the Examination and Processing of Human Semen, the goal of the manual is to meet the growing needs for the standardization of semen analysis procedures. With constant advances in andrology and reproductive medicine and the advent of sophisticated assisted reproductive technologies for the treatment of infertility, the manual has been continuously updated to meet the need for new, evidence-based, validated tests to not only measure semen and sperm variables but also to provide a functional assessment of spermatozoa. The sixth edition of the WHO manual, launched in 2021, can be freely downloaded from the WHO website, with the hope of gaining wide acceptance and utilization as the essential source of the latest, evidence-based information for laboratory procedures required for the assessment of male reproductive function and health. As stated clearly in all editions of the WHO Laboratory Manual for the Examination and Processing of Human Semen, the goal of the manual is to meet the growing needs for the standardization of semen analysis procedures. With constant advances in andrology and reproductive medicine and the advent of sophisticated assisted reproductive technologies for the treatment of infertility, the manual has been continuously updated to meet the need for new, evidence-based, validated tests to not only measure semen and sperm variables but also to provide a functional assessment of spermatozoa. The sixth edition of the WHO manual, launched in 2021, can be freely downloaded from the WHO website, with the hope of gaining wide acceptance and utilization as the essential source of the latest, evidence-based information for laboratory procedures required for the assessment of male reproductive function and health. DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/34279 DIALOG: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/34279 The World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen was first published in 1980 to standardize the procedures for the examination of human semen. This WHO “semen” manual has undergone five revisions (Fig. 1) and has been translated into multiple languages in response to the growing global needs in andrology and reproductive medicine and to standardize procedures for the examination of human semen. The WHO manual provides standard laboratory methods for semen analysis that are used extensively by clinical and reference laboratories for the understanding of reproductive function in men, the diagnosis and planning of treatment for subfertile couples, assessment of male contraceptive methods, and large-scale population studies and research activities, such as the effects of viral, environmental, and other toxicants on male reproductive function. The development of each edition of the manual was coordinated by the United Nations Development Programme/United Nations Population Fund/United Nations Children's Fund/WHO/World Bank Special Programme of Research, Development and Research Training in Human Reproduction (HRP) within the WHO Department of Reproductive Health and Research, which worked with the respective Task Forces or later, Research Group for the Regulation of Male Fertility. The latter, including the editorial board, was drawn from global experts working in male reproductive function. HRP has played very vital roles in convening the development of the manual, including overall coordination, technical input, supporting evidence generation, document production, dissemination, and promotion, ensuring continuity across editions, and providing necessary financial support. Between 1976 and 1977, the WHO Task Force on Methods for the Regulation of Male Fertility (led by C.A. Paulsen, M.D., University of Washington, Seattle, and M.R.R. Prasad, Ph.D., Manager of the Task Force) recognized the importance of standardizing semen analysis to improve the quality of results allowing for the exchange and combination of data across laboratories. A series of consultations in Barcelona, Geneva, Berlin, and Hong Kong were held to develop a laboratory manual for semen analysis. These consultations/workgroups were conducted in collaboration with two other WHO Task Forces on the Diagnosis and Treatment of Male Infertility and Vaginal and Cervical Devices for Fertility Regulation within the HRP from the first to the current (sixth) edition. The support of the respective HRP directors and scientists has been critical in the development and publication of the manuals. The first edition, called the Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction, was based on these consultations with 33 participants from Asia, Australia, Europe, North America, and South America. The focus of the manual was on semen analysis as the first laboratory test to study the physio-pathological testicular function in adults and establishing male fertility status in infertile couples, as well as monitoring spermatogenesis during and after male fertility regulation. The main objective of the first edition was to provide laboratory procedures for semen analysis that are standardized, precise, reproducible, sensitive, and validated. The manual consisted of only 43 pages with sections on the collection of the semen sample, initial examination, and assessment of sperm motility, concentration, morphology (including plates showing normal and abnormal spermatozoa), and viability. It included an Appendix with morphological classification and Papanicolaou staining for human spermatozoa with a standardized example for a semen analysis report form. It also included a section on sperm-cervical mucus interaction as a surrogate of sperm function, assessing sperm penetration of the cervical mucus using in vitro (capillary tube test) and in vivo (postcoital test) tests (1World Health OrganizationLaboratory manual for the examination of human semen and semen-cervical mucus interaction. Press Concern, Singapore1980Google Scholar) (Table 1).Table 1The major changes from the first to the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human SemenEditionYearPagesMajor changes1st198043Semen: sample collection, initial examination, sperm motility, sperm density, sperm morphology (plates and stains)Sperm-cervical mucus interaction: collection of mucus, in vitro test, postcoital test2nd198767Semen: Standard tests—includes all in the 1st edition + sperm antibody testsOptional tests—semen culture, seminal fluid biochemistry, zona-free hamster oocyte penetration test, sperm migration testCriteria of normality of semen samples3rd1992107Standard test—use “strict criteria for assessment of sperm morphology”Research tests—zona-free hamster oocyte penetration test, human zona pellucida binding test, acrosome reaction, computer-assisted sperm analysisSperm preparationQuality control of semen analysis4th1999128Optional tests—added hypoosmotic swelling test, multiple sperm defects indexResearch test—reactive oxygen speciesQuality control—statistical analyses of counting errors5th2010271Most extensive and comprehensive revision of the semen manualDetailed description of each procedureAdded total sperm output per ejaculate as a semen variableSperm motility combined rapid and slow into one grade of progressive motilitySperm preparations include spermatozoa from testis and epididymisUsing quality control to improve laboratory performanceAdded chapter on cryopreservation of spermatozoa6th2021276Step-by-step, easy-to-follow procedureBasic examination—standard tests, reintroduce slow progressive motilityExtended examination—optional tests included leucocyte, immature germ cells, added sperm aneuploidy, sperm genetics and DNA fragmentationAdvanced examination—research tests, added membrane ion channelsEmerging methods of semen analyses without a microscopeEliminated hamster zona-free penetration test, human zona binding test and section on sperm-cervical interaction Open table in a new tab The second, third, and fourth editions of the WHO semen manual were led by G.M.H. Waites, Ph.D., then as area Research Manager within HRP, in response to the developments in andrology, with increasing awareness of the importance of the objective assessment of the quality and functional characteristics of human spermatozoa (Table 1). More importantly, semen analysis was established as an important tool in assessing the male partner, with the rapid development of assisted reproductive techniques. A working group of experts in semen analysis, andrology, and reproductive medicine were constituted to revise the semen manual. The second edition was published in 1987 (2World Health OrganizationWHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction.2nd ed. Cambridge University Press, Cambridge1987Google Scholar). Among the included changes from the first edition was the division of semen analysis into standard (all tests described in the 1980 manual plus antisperm antibody tests) and optional tests, including semen culture, seminal fluid biochemical tests, zona-free hamster oocyte penetration test, and sperm migration test (“swim-up”). The section on semen-cervical mucus interaction was not changed. Importantly, the second edition introduced the “normal” values for sperm variables based mainly on the early studies of Macleod and Gold in 1951 (3Macleod J. Gold R.Z. The male factor in fertility and infertility. II. Spermatozoon counts in 1000 men of known fertility and in 1000 cases of infertile marriage.J Urol. 1951; 66: 436-449Google Scholar, 4Macleod J. Gold R.Z. The male factor in fertility and infertility. III. An analysis of motile activity in the spermatozoa of 1000 fertile men and 1000 men in infertile marriage.Fertil Steril. 1951; 2: 187-204Google Scholar, 5MacLeod J. Gold R.Z. The male factor in fertility and infertility. IV. Sperm morphology in fertile and infertile marriage.Fertil Steril. 1951; 2: 394-414Google Scholar), which was updated in 1979 (6MacLeod J. Male fertility in of semen a of the a study of the Steril. Scholar) with The manual that these values from such large-scale studies be used by laboratories to the of semen analysis was that the data of Macleod (3Macleod J. Gold R.Z. The male factor in fertility and infertility. II. Spermatozoon counts in 1000 men of known fertility and in 1000 cases of infertile marriage.J Urol. 1951; 66: 436-449Google Scholar, 4Macleod J. Gold R.Z. The male factor in fertility and infertility. III. An analysis of motile activity in the spermatozoa of 1000 fertile men and 1000 men in infertile marriage.Fertil Steril. 1951; 2: 187-204Google Scholar, 5MacLeod J. Gold R.Z. The male factor in fertility and infertility. IV. Sperm morphology in fertile and infertile marriage.Fertil Steril. 1951; 2: 394-414Google Scholar, J. Male fertility in of semen a of the a study of the Steril. Scholar) were from of fertile and infertile men, the to a in the was not The manual that be from men with within (Table were to provide technical of of the procedures. This 1987 translated into was used in WHO that were held in Asia, and with a of semen analysis in from the second to the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human sperm motility from (3Macleod J. Gold R.Z. The male factor in fertility and infertility. II. Spermatozoon counts in 1000 men of known fertility and in 1000 cases of infertile marriage.J Urol. 1951; 66: 436-449Google Scholar, 4Macleod J. Gold R.Z. The male factor in fertility and infertility. III. An analysis of motile activity in the spermatozoa of 1000 fertile men and 1000 men in infertile marriage.Fertil Steril. 1951; 2: 187-204Google Scholar, 5MacLeod J. Gold R.Z. The male factor in fertility and infertility. IV. Sperm morphology in fertile and infertile marriage.Fertil Steril. 1951; 2: 394-414Google Scholar), data from men with known fertility and men in infertile as used for normal sperm as studies of normal in data from assisted reproductive normal be with in vitro values of fertile men reference studies of fertile men as men within after J. Health Organization reference values for human semen using criteria of abnormal sperm morphology in in vitro Steril. Scholar, of criteria for of sperm of semen variables from fertile men studies of fertile men as men a with a to of of semen examination results a of data for the WHO semen analysis manual using criteria of abnormal sperm morphology in in vitro Steril. Scholar, of criteria for of sperm Scholar) Open table in a new tab the rapid in assisted reproductive used for the treatment of the infertile the of andrology laboratories in treatment the increasing the effects or on male reproductive function, and the need for new and standard methods of semen analysis, the WHO laboratory manual was by a group of experts in Health OrganizationWHO Laboratory manual for the examination of human semen and sperm-cervical mucus ed. Cambridge University Press, Scholar). The semen analysis section was into sections standard optional tests, and research The research tests included the zona-free hamster oocyte penetration test, human zona pellucida binding tests, acrosome reaction, and computer-assisted sperm analysis. The section on sperm-cervical mucus interaction was not but the of this edition was to the WHO Laboratory Manual for the Examination of Human Semen and Mucus This edition two to the needs of sperm and quality control of laboratory of semen in the this manual introduced and for laboratory semen analysis. These were and to the needs of laboratories. the the revision of the edition of the manual, the classification of sperm morphology was based on the morphology of spermatozoa to the oocyte and to the zona evidence to in in vitro that in the were normal sperm morphology was of abnormal sperm morphology in in vitro Steril. Scholar, as a factor in in vitro Steril. Scholar). Appendix the reference of semen variables were based on in the first and second It was that were clinical studies on semen variables at that and an reference for sperm morphology in men was as without data Health OrganizationWHO Laboratory manual for the examination of human semen and sperm-cervical mucus ed. 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