Introduction

Male infertility is defined as a failure of conception after one year of unprotected sexual intercourse (1). The global prevalence of male infertility ranges between 2.5% and 12% (Reference Thonneau, Marchand, Tallec, Ferial, Ducot and Lansac2, Reference Vander Borght and Wyns3) with a higher prevalence in Central/Eastern European countries and Australia (Reference Agarwal, Mulgund, Hamada and Chyatte4). The evaluation of male infertility includes a detailed medical history, drug and environmental exposure assessment, as well as reproductive and sexual history. Semen analysis remains the fundamental diagnostic test for the infertile male (Reference Leslie, Soon-Sutton and Khan5).

Semen analysis provides critical information regarding the contribution of the male factor in an infertile couple. It assists in the evaluation of opportunity for natural conception, time to pregnancy, and management of couples seeking assisted reproductive technology (ART) (Reference Buck Louis, Sundaram, Schisterman, Sweeney, Lynch and Kim6, Reference Pfeifer, Butts, Dumesic, Fossum, Gracia and La Barbera7). The semen analysis also helps in the identification of potentially reversible medical conditions such as accessory sex gland infections and epididymo-orchitis, which can impact fertility, and in decision-making for the level of ART when there is no correctable etiology (Reference Jungwirth, Giwercman, Tournaye, Diemer, Kopa and Dohle8).

The World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen is a technical guide for performing of a standardized semen analysis; the sixth edition was published in 2021 (1). While primarily a laboratory handbook, it also seeks to provide clinicians with an understanding of the various aspects of semen analysis and to serve a reference for researchers in male fertility. However, it was not designed to be a clinical manual, and, as a result, most clinicians refer to the WHO Manual only for the reference range to interpret the semen report as fertile or infertile. They ignore all the other tests in the manual because the WHO Manual does provide the clinical interpretation of these tests. Therefore, clinicians often limit their use of the semen analysis to basic parameters, neglecting the more detailed or advanced tests.

The WHO Manual’s scope covers much more than just sperm count, motility, and morphology. There are many other basic parameters that have clinical relevance. The manual also describes many extended and advanced tests that can be of significance in selected cases. Further, the sixth edition of the WHO Manual has taken the bold step of abolishing reference ranges traditionally used to distinguish between fertile and infertile semen samples. This change requires an understanding of the proper interpretation of the semen report to ensure that the manual remains clinically relevant. If clinicians do not fully understand the test or its clinical implications, then they may misinterpret the results. As a result, the comprehensive diagnostic value of the WHO Manual could be undermined.

Semen analysis is the primary tool in male infertility assessment. A properly done semen analysis is critical in establishing the diagnosis of azoospermia (Reference Aziz9). Azoospermia is observed in up to 2% of adult men and 9% of infertile men, of which 6% have azoospermia due to spermatogenic failure and 3% due to ductal obstruction (Reference Thonneau, Marchand, Tallec, Ferial, Ducot and Lansac2). In cases of azoospermia, the ejaculate volume and the pH measurements are useful tools in differentiating the etiology (Reference Aziz9). The book will discuss other associated findings that a clinician should look at in a report with low semen volume, such as the pH and the seminal fructose levels. Furthermore, it will guide the reader towards the workup that helps in establishing the differential diagnosis (see Clinical Application 1.1 and Clinical/Technical Application 1.2).

Semen analysis may serve other purposes, and poor semen parameters have been associated with fetal malnutrition in utero and early life, increased risk of cardiovascular disease and diabetes mellitus, and may potentiate other adverse health outcomes in adult life (Reference Barker15, Reference Omu16). The mechanisms by which early-life conditions are associated with male reproductive disorders and major late-life diseases are still not well understood (Reference Omu16). In this book, the mechanisms related to these effects will be explored with a special emphasis on modifiable risk factors, metabolic syndrome, and androgen deficiency.

Why Is a Standardized Laboratory Manual Needed?

Semen analysis is the cornerstone of laboratory evaluation in the assessment of male infertility, providing essential information to determine the presence and severity of male-factor infertility within a couple (Reference Schlegel, Sigman, Collura, De Jonge, Eisenberg and Lamb17). It can diagnose infertility in men who have azoospermia, severe asthenozoospermia, or monomorphic sperm defects like globozoospermia, but has a limited role in the prediction of overall infertility since female factors can influence fecundity in most couples (Reference Patel, Leong and Ramasamy18). Furthermore, while the threshold values for sperm concentration, motility, and morphology can be used to classify men as subfertile, of indeterminate fertility, or fertile, none of the measures are diagnostic for infertility (Reference Guzick, Overstreet, Factor-Litvak, Brazil, Nakajima and Coutifaris19). Sperm motility and concentration seem to provide more accurate information than morphology regarding a man’s fertility potential (Reference Nallella, Sharma, Aziz and Agarwal20).

The understanding of the principles and procedures for the collection and analysis of semen specimens, the reference ranges for normal semen parameters, and definitions of various categories of abnormal semen parameters play a significant role in clinical practice and in the management of infertile patients (Reference Agarwal, Sharma, Gupta, Finelli, Parekh and Selvam21). However, the standardization of semen analysis parameters is very difficult for many reasons, including inherent variability in semen parameters, the use of subjective techniques with no standards for comparison, poor technician training, deficiencies with proficiency testing, and a reluctance to change techniques (Reference Brazil22).

To date, the WHO has issued six manuals for standardizing the semen analysis (1). An earlier study in the United Kingdom showed that very few laboratories (5%) were compliant with WHO guidelines regarding morphology assessment, including methods of staining and observation, classification and sampling methods, and participation in internal and external quality control programs (Reference Riddell, Pacey and Whittington23). Similarly, another study in the United States very few laboratories performed quality control for sperm counts, motility, and morphology, and there were significant variations in the semen analysis assessment (Reference Keel, Stembridge, Pineda and Serafy24). Additionally, there is a lack of trusted, quantitative, and predictive diagnostic tests that look inside the sperm to quantify defects such as DNA damage, RNA abnormalities, centriole dysfunction, or reactive oxygen species to discover the underlying cause (Reference Pandruvada, Royfman, Shah, Sindhwani, Dupree and Schon25).

The Sixth Edition of the WHO Laboratory Manual

The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen replaces the 2010 fifth edition. The sixth edition provides important information on various tests for semen examination and assessment, sperm cryopreservation, and quality control in the andrology laboratory (1). The manual aims at facilitating precise compliance with the recommended procedures with step-by-step instructions that are easier to follow, and with fewer variants in dilutions and calculations (Reference Barker15, Reference Chung, Arafa, Boitrelle, Kandil, Renkel and Saleh26).

Although the principles for basic semen examination remain mainly largely unchanged in the sixth edition, important modifications have been made to improve efficacy, compliance with basic laboratory science, and user-friendly instructions. For human sperm morphology assessment, more rational techniques are given for the assessment of defects in all parts of the spermatozoa (Reference Barker15). One of the strengths of this new edition is a significant expansion of the normative data by including new geographical areas, and the inclusion of roles for Sperm DNA Fragmentation (SDF) testing and fluorescent in situ hybridization (FISH) assays, as well as other extended tests in certain situations (Reference Barker15, Reference Paffoni, Somigliana, Boeri and Viganò27).

While formulating the reference values for semen assessment in the sixth edition, three key challenges were identified, namely the quality of semen assessments, the transparency and reporting of data, and, lastly, the paucity of studies with primary data (Reference Wang, Mbizvo, Festin, Björndahl and Toskin28). Further, methodological concerns have been raised (Reference Paffoni, Somigliana, Boeri and Viganò27), and more studies are needed to clarify whether the abandoning of reference values in the sixth edition represents an unquestionable choice.

Although the above factors can limit the laboratory assessment of male infertility, this field has witnessed several significant advances in recognition of the key role that the male plays in human fecundity, and advances in biotechnology and a new generation of tests of sperm function are being developed to help us understand the various pathophysiological mechanisms in testicular dysfunction (Reference Gül, Russo, Kandil, Boitrelle, Saleh and Chung29). Therefore, semen analysis should be complemented with other sperm functional assays, to indirectly measure the ability of the spermatozoa to deliver the correct complement of chromosomes to the ovum for successful conception (Reference Agarwal, Roychoudhury, Bjugstad, Cho, Agarwal, Parekh and Aziz30).

There is a need to develop accurate, automated methods for semen analysis and for testing sperm functional potential. This could include the development of point-of-care devices that have been fully validated and include accompanying quality assessment tools (Reference Tsao, Yang, Wen, Chang, Matsuura and Chen31). Overall, a multiparameter evaluation of the same cells could render more accurate estimates of the capacity of spermatozoa to initiate fertilization and support embryonic development. Efforts in this direction are represented by image flow cytometry, a tool that would allow assessments of high numbers of cells and locate where changes are taking place. Next-generation computerized semen analysis systems may also facilitate this multiparametric analysis, although through a different approach (32).

Finally, the WHO Manual, sixth edition, recognizes that andrological management decisions are multifaceted and cannot be limited to the use of sperm analysis and 5th percentile values. For this reason, they have eliminated the concept of “norms,” and all pathological terms (like necrozoospermia, asthenozoospermia) have been deleted in the sixth edition. It is clear that the manual is a technical guide for the standardizing semen analysis. However, since 2021, the andrologist–clinician has been faced with a paradox: On the one hand, the WHO Manual is robust, technically rigorous, and precise; but, on the other hand, the clinician who wishes to use the semen analysis to guide clinical decisions no longer has defined reference values. In the absence of thresholds, it is difficult for the clinician to reach a diagnosis and guide further evaluation and therapy. This book discusses this subject in detail and proposes approaches to interpret the semen test from a clinical point of view.

Conclusion

Given the clinical limitations of the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, this book, written by authors affiliated with the Global Andrology Forum (Reference El Ansari, Savira, Atmoko, Shah, Boitrelle and Agarwal33, Reference Pinggera, Ramsay, Zini, Atmoko and Colpi34), has been structured as a comprehensive clinical companion to the technical WHO manual on semen examination. This book covers test indications, clinical utility, data interpretation, decision-making algorithms, and cost-effectiveness. It is the product of collaborative inputs from experts in reproductive medicine, andrology, and laboratory medicine. It incorporates the latest guidelines and research in the field to ensure that the clinical recommendations in the manual reflect the most current knowledge.