Male infertility is a common condition that makes a significant contribution to demographic problems that are extremely characteristic of modern Russia [1–4]. A semen analysis is the main method at the initial diagnosis stage of male infertility. This study allows to assess the male reproductive potential, as well as to determine the most effective treatment tactics for a married couple. Over the past 40 years, WHO experts have been trying to standardize the methodology of ejaculate research. The first edition of the WHO guidelines for the study and treatment of human ejaculate was published in 1980. Since then, five updates of this manual have been published, each of them attempts to interpret the statistics available at the time of publication to separate the population of fertile and infertile men. Each new manual analyzes considers and corrects the shortcomings of the previous editions.

In March 2021, the WHO released a preliminary version of the sixth edition for public discussion and proposals [5]. In fact, the new manual consists of three parts: sperm examination, preparation and cryopreservation of ejaculate, quality control, and assurance. The sections concerning the sperm examination procedure include basic (routine) studies, additional studies (which can be used by laboratories or clinicians in certain situations) and so-called “advanced” (progressive) tests (which are currently not recommended for routine use and are intended mainly for research purposes). In the sixth edition, the procedure for performing the study is described in detail and in stages for the technique to be accurately reproduced in other laboratories. Extended and in-depth examination sections have been completely redesigned according to current clinical practise.

In this article, the authors consider the main changes made to the Sixth Edition of the WHO laboratory manual for the examination and processing of human semen, as well as their advantages and disadvantages, and their impact on current clinical practise.

One of the most significant changes proposed in the updated version of the WHO guidelines is the proposal to abandon reference intervals, since they do not allow for reliable differentiation of fertile and infertile patients. The reference intervals adopted in the fifth edition of WHO were based on data from population studies involving 1800 fertile patients [6]. The 5th percentile was taken as a reference value in the fifth edition. The decision to adopt such a lower threshold value was criticised because it did not allow unambiguously differentiating infertile and fertile men [7]. Several studies have also noted that the use of reference values adopted in the fifth edition, instead of those established in the fourth edition, leads to the fact that between 15% and 44% of men with reduced fertility will have normozoospermia [8–11]. For this reason, many specialists in the field of reproductive medicine believe that the thresholds adopted in the fifth edition do not adequately reflect the fertility potential of many patients observed in reproduction clinics.

It is necessary to note that there are not any reference intervals, and probably they cannot exist, as they would completely solve the problem of infertility diagnosis and separation of uniquely infertile from fertile individuals.

In this regard, in the sixth edition, a proposal is put forward to abandon the reference values and move to the so-called “decision limits”, which represent the division of the values of semen analysis indicators into ranges characteristic of fertile, infertile, as well as the allocation of the area of boundary values – the gray zone. It is proposed to consider the occurrence of the semen analysis values of a particular patient in a certain range as a guideline for the clinicians regarding the tactics of diagnosis and treatment. In fact, this is why these ranges were called 'decision limits'. It is important to remark that this approach is not new. In 2001, Guzick et al. proposed the concept of two levels of reference values with the allocation of a 'grey zone'. They examined the results of at least two semen analyses of 765 infertile patients and 696 fertile ones. The spouses of the men who participated in the survey did not have a female factor of infertility. According to the study data, the indicators of sperm concentration less than 13.5 × 106/ml, mobility less than 32%, and normal morphology less than 9% were determined in subfertile men. The fertile patient group had sperm concentrations of more than 48.0 × 106/ml, mobility – more than 63% and normal morphology – more than 12%. Indicators in the interval between these two levels of values, thus, fall into the gray zone [12].

Of course, the application of decision-making limits would theoretically make it possible to make a more correct judgment about the clinical situation, since it would introduce a biologically more correct understanding of the norm regarding the fertility of the subjects.

The adoption of a grey or borderline zone would probably have a significant effect on the clinical practise of andrologists, since many patients, instead of immediately being referred for in vitro fertilisation due to idiopathic infertility, are now likely to be in the grey zone and will be treated as patients with decreased fertility. For this reason, they will be referred to an andrologist and examined more thoroughly, which, of course, may possibly increase the effectiveness of the infertility treatment in a couple.

However, the abolition of reference values will also lead to the abolition of such terms as oligo-, astheno- and teratozoospermia. In this case, what should be the conclusion of the semen analysis? What ranges of values should be accepted as the “decision limits” and based on what amount of data should they be developed?

Nowadays, there is not any certain answer to these questions.

Despite the 'call' to abandon the reference values, in the sixth edition, WHO experts recalculated the fifth percentile based on updated data and research results were published before 2020. The sixth edition includes data from 5 new studies, from 2 regions of Europe and Asia, as well as one Africa region. However, it should be noted that most patients in the Asian region are represented by residents of China, which is certainly a specific factor, as data on the Chinese population can ambiguously reflect the characteristics of the population of other regions of Asia. In addition, this guide is criticised for the lack of studies on the population of Latin American countries [13].

Changes in the mobility assessment system deserve a separate discussion. The fifth edition was criticized for the decision to remove the assessment of progressive mobility by category. In the sixth edition, one can see the presence of these categories again: rapidly progressively mobile spermatozoa moving at a speed of more than 25 microns/s, slowly progressively mobile spermatozoa moving at a speed of 5 to 25 microns/s, non-aggressively mobile spermatozoa (< 5 microns or less than one head length and stationery, no tail movement). This is indeed a very “expected” change, or rather, a return to the position of the fourth edition, since such a classification allows for a better assessment of such an important characteristic of sperm as motility, which in turn allows for a better overall assessment of fertility potential.

It is also necessary to mention the change in the position of WHO specialists on the threshold value for the percentage of spermatozoa bound with antisperm antibodies. Nowadays, there is no lower reference value to confirm the presence of the patient's immune factor of male infertility, which would meet the currently available standards of evidence-based medicine. In the fifth edition of the manual, a consensus opinion of experts was adopted concerning determining the threshold value for spermatozoa bound with antisperm antibodies at 50% [6]. However, the sixth edition of the WHO manual does not emphasise any specific reference value. Instead, it is proposed that each laboratory should determine its thresholds by testing a sufficiently large number of fertile men [5].

An important innovation also concerns the assessment of the integrity of the genetic material of spermatozoa. The editors of the sixth edition included the sperm DNA fragmentation test in the list of advanced methods. Techniques such as TUNEL, Comet, and SCD were described in detail, as well as notes on the clinical interpretation of these tests. As for the reference values, the authors of the sixth edition say that each laboratory should independently determine the reference values for each of the tests for assessing the integrity of the genetic material of spermatozoa based on practice. Of course, the assessment of the integrity of the genetic spermatozoa material is an important component of assessing the patient’s fertility potential. The authors of the sixth manual edition note that Comet analysis includes several stages, it has a high level of interlaboratory variability and requires a high level of laboratory assistant training; therefore, it may not be suitable for some laboratories.

The editors of the manual did not consider the clinical significance of these tests and did not provide any guidance regarding the indications for testing.

The authors of the sixth edition emphasised the importance of studying the level of oxidative stress and devoted a separate subsection to this study. Probably, due to the limited availability of methods to determine active radicals, the subsection devoted to this problem was included in the section on “advanced” methods and should still be considered to a greater extent as a method used in scientific research. However, given the large number of published studies indicating the effect of excessive production of reactive oxygen species on male fertility, as well as the isolation by many authors of male infertility caused by oxidative stress as a separate clinical entity [14], the study of the level of oxidative stress can be considered in separate clinical situations.

The series of editions of the WHO guidelines on the study and processing of human ejaculate certainly reflects the evolution of ideas about the assessment of male fertility. The authors of the new manual considered and corrected the shortcomings of previous editions. Some concepts, which were adopted earlier and then abolished, were revised again, and returned to the pages of the manual. Despite innovations, many problems related to the complexity of assessing male fertility remain unresolved.