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

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.

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Goals of the WHO laboratory manual for examination of human semen

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.

Figure 1

The six editions of the WHO “Semen” manual.

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.

The beginning

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 (1) (Table 1).

Table 1

The major changes from the first to the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen

Edition	Year	Pages	Major changes
1st	1980	43	Semen: sample collection, initial examination, sperm motility, sperm density, sperm morphology (plates and stains)Sperm-cervical mucus interaction: collection of mucus, in vitro test, postcoital test
2nd	1987	67	Semen: 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 samples
3rd	1992	107	Standard 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 analysis
4th	1999	128	Optional tests—added hypoosmotic swelling test, multiple sperm defects indexResearch test—reactive oxygen speciesQuality control—statistical analyses of counting errors
5th	2010	271	Most 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 spermatozoa
6th	2021	276	Step-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

The second edition

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 (2). 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 (3, 4, 5), which was updated in 1979 (6) with consensus-based modifications. The manual suggested that these values from such large-scale studies may be used by laboratories to guide the interpretation of semen analysis results. However, it was noted that although the data of Macleod et al. (3, 4, 5, 6) were obtained from large numbers of fertile and infertile men, the time taken to achieve a pregnancy in the female partner was not defined. The manual suggested that “normal values” should be evaluated from men who had achieved pregnancy with their partner within 12 months (Table 2). Appendices were expanded to provide technical details of some of the procedures. This 1987 version, translated into seven languages, was used in WHO Andrology Workshops that were held in Africa, Asia, and Eastern Europe with a hands-on demonstration of semen analysis techniques.

Table 2

Changes in Reference Values from the second to the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen

Edition	Nomenclature	Semen volume (ml)	Sperm concentration (106/ml)/Total no. (106/ejaculate)	Total /progressive sperm motility (%)	Normal forms (%)	Reference
2nd	Normal values	≥2	≥20/≥40	≥50/≥25	≥50	Modified from (3, 4, 5), data from 1,000 men with known fertility (female partner currently pregnant) and 1,000 men in infertile marriage
3rd	Normal values	≥2	≥20/≥40	≥50/≥25	≥30	Same as 2nd EditionEmpirical value used for normal sperm forms
4th	Reference values	≥2	≥20/≥40	≥50/≥25	∗	Same as 3rd Edition. ∗ Normal forms—population-based studies of normal forms in progress, data from assisted reproductive technology suggest <15 normal forms may be associated with decreased in vitro fertilization rate
5th	Reference values of fertile men (5th centile lower reference limits)	1.5	15/39	40/32	4	Population-based studies of approximately 1,900 fertile men (defined as men whose partner conceived within 12 months after stopping contraception) (23).Morphology using “strict” criteria (8, 11)
6th	Distribution of semen variables from fertile men (5th centile)	1.4	16/39	42/30	4	Population-based studies of approximately 3,500 fertile men (defined as men whose partner had a natural conception with a confirmed time to pregnancy of ≤12 months) (41).Morphology using “strict” criteria (8, 11)

The third edition

Recognizing the rapid progress in assisted reproductive techniques used for the treatment of the infertile couple, the proliferation of many andrology laboratories in infertility treatment centers, the increasing concern about the putative effects or environmental pollutants on male reproductive function, and the need for new and better standard methods of semen analysis, the WHO laboratory manual was revised by a group of experts in 1992 (7). The semen analysis section was divided into three sections standard procedures, optional tests, and research tests. 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 significantly altered, but the title of this edition was changed to the WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. This edition contains two additional chapters relevant to the needs of laboratories, sperm preparation techniques and quality control of laboratory performance of semen analyses. In addition, in the appendices, this manual introduced basic safety guidelines and minimal equipment needed for any laboratory performing semen analysis. These additions were timely and responsive to the needs of many laboratories.

In the few years preceding the revision of the third edition of the manual, the classification of sperm morphology was changed based on the morphology of spermatozoa able to reach the oocyte and bind to the zona pellucida. Emerging evidence related to outcomes in in vitro fertilization suggested that pregnancy rates in the female partner were more frequent when normal sperm morphology was above 14% (8, 9). In Appendix 1, the reference limits of semen variables were provided based on consensus limits in the first and second editions. It was noted that there were no population-based clinical studies on semen variables completed at that time, and an empirical reference value for sperm morphology in men was quoted as 30% without supportive data (7) (Table 2). This empirical cut-point of 30% normal forms was reported not to be clinically useful as approximately half of fertile men had sperm morphology below this value (10). It is important to keep in mind that the criteria for characterizing human sperm morphology have changed substantially from those used by Macleod (5) to the strict Tygerberg criteria (11).

The fourth edition

In 1999, the WHO/HRP fourth edition of the semen manual was published (12). This revision was in response to advances in the genetics of male infertility (Y chromosome microdeletions) (13); the success of intracytoplasmic sperm injection (14); the demonstration of contraceptive efficacy of hormonally induced suppression of spermatogenesis (15, 16); and reports of declining sperm counts (17) and increasing incidence of testicular cancer (18). These factors added urgency to the search for new methods to achieve better standardization and improved procedures for semen analysis. The WHO sought advice from experts throughout the world via letters, journals, and national andrology societies and formed a working group to revise the manual. The fourth edition of the WHO semen manual was a companion to the WHO Manual for the Standardized Investigation and Diagnosis of the Infertile Couple (19). In this fourth edition, the multiple sperm defects index and the hypoosmotic swelling test were added to the optional tests, and measurement of reactive oxygen species was added to the research test. Importantly, the quality control section was expanded to include statistical consideration of counting errors. Again, in Appendix—“Reference Values for Semen Variables”—it was clearly stated that these reference values are not to be used as the minimum/lowest semen values compatible with fertility in vitro or in vivo. Taking into consideration the comments from the prior edition, this edition left the morphology variable blank with a note suggesting that a percent normal morphology <15% may be associated with a lower in vitro fertilization rate (Table 2). This fourth edition of the WHO Semen Manual was considered a significant improvement in the description and standardization of techniques, and the demand for the manual was high (20, 21).

The expanded fifth edition

After reviewing prior editions, WHO/HRP, in consultation with its Research Group for Male Fertility Regulation, the fifth edition of the laboratory manual’s name was changed to WHO Laboratory Manual for the Examination and Processing of Human Semen, and the publisher changed from Cambridge University Press to the World Health Organization (22). The manual thus became free, available in print, and as an electronic version downloadable from the WHO website (https://apps.who.int/iris/handle/10665/44261), in an attempt to establish the manual as a global guidance that will set standards across various laboratory settings and resources. This increased the demand even further, with an external evaluation of HRP noting that the manual was among its global publications with the highest citations (4,271) and downloads (53,863 in the past 6 months). It also received further requests for translations. The fifth edition was the most extensive and comprehensive edition of the manual with 271 pages.

Ten years after the fourth edition, the WHO considered revisions to the manual necessary because of advances in andrology technology and new tests that may be useful in the management of infertile couples with updated supporting evidence. After consultations with experts in the field and professional associations, the WHO/HRP, led then by Research Manager and later Director, M. Mbizvo, Ph.D., established an editorial committee with 20 members from Africa, Asia, Australia, Europe, and North America. The committee nominated T. G. Cooper, Ph.D. from the Institute of Reproductive Medicine at the University of Münster, Münster, Germany, a WHO-collaboration center, as editor-in-chief with support from the WHO Secretariat. After receiving comments from laboratory technicians and scientists, this edition improved on the prior editions and provided a detailed description with a rationale of alternate methods, examples, notes, and comments for each procedure. Dr. Cooper and the editorial committee decided to edit the fifth edition in accordance with the principles of “Evidence Based Medicine.” This led the editors to collect and critically appraise original clinical studies in the field of spermatology and the definition of evidence-based thresholds and reference values. Semen analyses procedures remained the same, but the emphasis was placed on counting errors when an inadequate number of spermatozoa are assessed. Sperm preparation techniques extended beyond the ejaculate and included the collection of spermatozoa from the testis and epididymis. A chapter on cryopreservation of spermatozoa was added. The quality control chapter was rewritten with suggestions on how to improve laboratory performance. Most importantly, the fifth edition for the first time provided reference ranges based on in vivo data derived from studies in fertile men with partners who conceived within 12 months (23). The lower fifth centile was used as the lower reference range with a clear statement indicating that this lower limit does not distinguish subfertile and fertile men and that clinical information must be used together with semen analyses data (Table 2).

Although this edition of the WHO semen manual became one of the most frequently downloaded publications of the WHO/HRP, the reviews of the edition continued to be very favorable. Comments were received from all over the world on the usefulness of this edition in clinical and research settings (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). Of note, some scientists commented that the wide use of assisted reproductive technology using intracytoplasmic sperm injection often bypassed in-depth evaluation of the male partner. In some men, understanding the qualitative and functional deficiency of spermatozoa including poor motility (e.g., primary ciliary dyskinesia), low viability (e.g., damage by reactive oxygen species), gross morphological abnormality (e.g., globozoospermia, macrozoospermia, different abnormalities of sperm flagella), allowed couples to consider further sperm functional assessment and genetic tests before making informed decisions on the optimal method of treatment (34, 35). Importantly, traditional semen parameters in at least a single ejaculate are considered necessary for the appropriate investigation and treatment pathway for couples with male infertility (36). Comments also suggested that this laboratory manual should follow the standardization in describing tests in clinical laboratories according to the International Federation of Clinical Chemistry and Laboratory Medicine and the International Union of Pure and Applied Chemistry (37) as well as the International Organization for Standardization.

Using the WHO laboratory manual to disseminate information and improve standardization

In the years between 1987 and 1999, the WHO/HRP sponsored many Andrology workshops in Africa, Asia, and Eastern Europe to introduce the standard techniques of semen analyses to many countries. Many of the centers were participating in studies sponsored by the Task Force on Methods for the Regulation of Male Fertility, which later became the Research Group on Male Fertility Regulation. The WHO manuals continued to be translated into many languages by individuals from national andrology societies (Arabic, Chinese, French, German, Greek, Indonesian, Italian, Japanese, Portuguese, Turkish, and Russian). This enabled laboratory technicians who were not proficient in English to be able to use the manual.

Most importantly, national and international societies such as the American Society of Andrology and the European Society for Human Reproduction and Embryology (ESHRE) held many basic semen analysis courses (38) using the various versions of the WHO laboratory manual as the teaching guide. In the African region, semen analysis and andrology workshops were funded by HRP, using their Research Capacity Strengthening grants, and hosted by T. Kruger, M.D., and his group in Cape Town, South Africa. Based on 20 ESHRE Basic Semen Analysis courses, these structured training courses led to a substantial reduction of between observer variability in semen analyses (39). A recent publication showed that the participation of external quality control programs reduced interobserver variability of sperm concentration, but the participation of the ESHRE Courses reduced variability not only for sperm concentration but for sperm motility and morphology (40).

The effectiveness of the WHO laboratory manual in improving global standardization is exemplified in large, population-based studies of healthy men where 5 of the 32 studies used only the WHO laboratory manual-recommended hemocytometer for assessing sperm concentration (23). In the follow-up data, four of the five additional studies used the same hemocytometer for assessing sperm concentration (41). These data were derived from laboratories in five continents that reported compliance with the WHO laboratory manual. Racial–ethnic differences in semen variables were not reported by these analyses, but there is a paucity of studies from Asia and Africa and none from South America. Regional differences in semen quality have been reported in men from Europe (42) and the United States (43). The authors suggest that the regional difference in semen quality may be related to environmental exposures, different lifestyles, and agricultural vs. urban areas.

Methodology for preparation of the sixth edition

The sixth edition of the manual includes a section that describes the methodology used by WHO to prepare this sixth edition, providing transparency and assuring equity of representation as well as technical accuracy according to the WHO requirements. The WHO/HRP secretariat used the list of members and contributors from the prior editorial team, consulted with the collaborating institutions, and scanned the peer-reviewed global literature on andrology and semen analyses to develop the final list. This final list of editorial committee members selected for the editorial committee had a record of accomplishment of research and practice on semen analysis, balancing laboratory experience and clinical practice. The editorial committee consisted of nine international members, two members from the WHO Secretariat, and three early-career scientists. This manual retained its purpose as a laboratory procedure document and was not considered as a guideline, as the objectives and procedures for the latter are very different. This editorial team reviewed the topics, assigned members to review the literature on special topics that may be more controversial, and the reviews were discussed and a decision was made based on the available evidence whenever possible.

The procedure undertaken took approximately 3.5 years with partial interruptions to timelines from the effect of the COVID-19 pandemic. The process entailed developing an outline of the chapters, preparing two major rounds of document drafts that were reviewed internally by the editorial core group, approving and reviewing by the editor-in-chief, Lars Björndahl, M.D., Ph.D., Centre for Andrology and Sexual Medicine, Karolinska Institutet, Stockholm, Sweden; internal reviewing by WHO; external reviewing by other technical experts on andrology and semen analysis, and public reviewing (March to June 2021) with comments from 43 countries. These steps are part of the standard procedures for the development and finalization of WHO documents after collation and synthesis of all reviews and comments that were carefully implemented within the development of the sixth edition. The procedures for the final launch of the manual followed the WHO best practice in this area.

The launch of the sixth edition in 2021

On July 20, 2021, the WHO launched the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen (https://www.who.int/publications/i/item/9789240030787) through a webinar. In the press release, the WHO states “High-quality laboratory services are crucial for ensuring the value of research on health. Effective analysis and comparison of results across laboratories help to ensure that health care workers receive high-quality information, which in turn helps them to provide the best quality care. The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen replaces the 2010 version. It provides important information on semen examination and preparation for clinical evaluation, assessment, cryopreservation, quality control in the semen analysis laboratory, and laboratory examination in the investigation of male sexual and reproductive health” (https://www.who.int/news/item/27-07-2021-who-launches-updated-manual-to-ensure-high-quality-testing-of-human-semen-in-clinical-and-research-settings).

Retaining the stated goals of the manual articulated with its first launch in 1980, this newest edition provides step-by-step, easy-to-follow procedures for technicians and scientists performing semen analyses. Consistent with previous editions, the sixth edition includes basic examination, semen volume, sperm motility, concentration, morphology, and vitality assessments as well as estimation of sperm numbers in samples with very low sperm concentration. Extended semen examination includes procedures to detect leucocytes, immature germ cells, sperm antibodies, indices of multiple sperm defects, and biochemical assessment and introduces methods to detect sperm aneuploidy, sperm genetics, and DNA fragmentation. Advanced examination replaced the prior research tests with extensive revisions and includes assessment of reactive oxygen species, membrane ion channels (new), acrosome reactions, and computer-assisted sperm analysis. Emerging new methods were briefly described, including tracking individual sperm cell motility and flagellar waveforms that may provide more information on the energetics of sperm movement. Continued improvement in digital camera technology in smartphones raises the possibility of initial semen assessment without a microscope. The zona-free hamster oocyte penetration test and human zona pellucida binding test are removed as these tests are rarely used in the laboratory because of difficulty in obtaining the test materials, lack of standardization, high labor intensity, and high cost. In addition, the availability of intracytoplasmic injection of sperm directly into oocytes eliminated the necessity of these tests. Chapters on sperm preparation and cryopreservation and internal and external quality control methods have been updated. The chapter on sperm interaction with cervical mucus has been eliminated, as the procedure is no longer being routinely performed as part of infertility investigation.

In the second, third, and fourth editions of the WHO manual, the consensus-based lower limits of semen variables were presented. At that time, population-based studies from men who have achieved conception within the prior 12 months were not available. The fifth edition reported WHO reference ranges in 2010 based on men whose partners achieved pregnancy within 12 months of trying to conceive (23). In the sixth edition, data from new publications from more diverse populations were amalgamated to the 2010 data; the results from approximately 3,500 fertile men from 12 countries in five continents validated and expanded the distribution of semen variables in the fifth edition (41). It was emphasized again that these distribution data with the fifth centile as the lower reference range do not define limits between fertile and subfertile men. There is a substantial overlap of semen evaluation between fertile and subfertile men (3), and there are no distinct boundaries between fertile vs. infertile men. The clinician may explain to the infertile couple that with the use of the distribution of semen variables in fertile men, for instance, the man’s sperm concentration/total number in the ejaculate is lower than 5% of fertile men (fifth centile sperm concentration <16 × 106/ml or total sperm number <39 × 106/ejaculate) (41) and then based on clinical assessment discuss the options of further investigation or treatment.

The manual in the final form can be downloaded using this URL: https://bit.ly/WHOSM6. It is anticipated that this edition of the manual will be a widely used teaching guide for semen analyses workshops and andrology training programs, for both basic and more advanced methods, adapting to the virtual meeting format that gained wide acceptance in the past year. Continued improvement in virtual meeting systems with a hands-on demonstration by the trainer followed by each course participant showing step-by-step procedures back to the trainer to allow guidance and further instructions. This may allow the training of clinicians, scientists, laboratory staff, and students to learn and adopt the standardized methods, which is the goal of the semen manual. However, it is essential to provide in-house performance training following the virtual sessions (44). Unlike the prior manuals, the sixth edition described only one recommended method of assessment of semen volume, sperm concentration, motility, and morphology which simplifies the recommended procedures for laboratories. The challenge is to implement external quality control to ascertain that the methods recommended by the WHO laboratory manual are implemented by laboratories to achieve standardization and reproducibility of results across laboratories around the globe (45). A further support to the global standardization is the recently published ISO standard (International Standards Organization; ISO 23162:2021 Basic semen examination—Specification and test methods, 2021, ISO, Geneva) based on the same principles and evidence base as the sixth edition.

Conclusion

The WHO Laboratory Manual for the Examination and Processing of Human Semen has undergone five revisions (Table 1) to meet the needs of laboratories performing semen analyses. Continuous improvement/updating of the manual occurs every 8 to 10 years to introduce new and emerging tests for research and clinical validation of significance into practice. The goals of this laboratory manual remain the same. It is designed for everyone who uses semen analyses to understand the function of the male reproductive function and dysfunction, diagnose and plan treatment of the infertile couple, assess sperm output and other variables following male contraception, and conduct epidemiological and other research studies of, among others, potential environmental pollutants and other toxicants on male reproductive function.