Sixth edition of the World Health Organization laboratory manual of semen analysis: Updates and essential takeaway for busy clinicians

Contributors: Dr. Vineet Malhotra (India), and Dr. Krishna C Mantravadi (India)

*Commentary:

Semen analysis (SA) is still considered the gold standard and is a preliminary step in a couple’s fertility journey. This basic screening of semen helps the clinical team plan further steps. However, while SA is the first investigation, it has several limitations and is not yet standardized enough to predict the time to conception or determine the best choice of assisted reproduction for overcoming infertility. Over the past few decades, scientists have developed six editions of the WHO manual to help standardize the evaluation of infertile men. The 6th edition, published in 2021, is the current version. In addition to basic semen analysis, it provides information on sperm preparation, cryopreservation, and quality control and assurance.

The new 6th Edition presents revised reference values for basic semen parameters. These values are based on data from fertile men in the 5th Edition, released in 2010, and five additional studies published between 2010 and 2020. This revision attempts to address a limitation in the 5th Edition, where reference values were skewed towards specific geolocations. However, the reference limits suggested by the WHO (World Health Organization 2021) come from a very mixed group of men and should not be mistaken for definitive limits between fertility and infertility. The new WHO manual emphasizes the development of decision limits, which are more significant than those from a mixed population.

A significant change from the previous 5th Edition is the abandonment of fixed reference values. The 6th Edition specifies that the 5th centile values are only one way to interpret SA results and that using the 5th centile alone is insufficient to diagnose male infertility.

The 6th Edition aims to optimize SA procedures by providing detailed steps and a methodological sequence for test execution. It also introduces new sperm tests for assessing sperm DNA fragmentation (SDF) and seminal oxidative stress (OS), while discontinuing obsolete tests like human cervical mucus evaluation.

Some adjustments have been made regarding basic semen parameters. The evaluation of semen odor has been added, noting that "urine or putrefactive odors may be of clinical interest," although standardizing this parameter is challenging due to its subjective nature.

 Regarding sperm motility, the 6th Edition re-adopts the distinction of progressive motility into two categories (grades a and b). This categorization, which includes fast progressively motile, slow progressively motile, non-progressively motile, and immotile (grades a, b, c, and d, respectively), was last used in the 4th Edition. It is surprising that this distinction is included in the 6th Edition without recent studies (post-2010) demonstrating its utility in andrology or routine diagnosis.

For evaluating sperm count, semen dilutions have been simplified, but 200 spermatozoa per replicate should be counted. In the past, observing 0–4 spermatozoa per field at ×400 magnification (or 0–16 spermatozoa per field at ×200 magnification) could provide enough indication for concentration assessment, with concentrations reported as less than 2 × 10^6/mL. This method has been revised in the 6th Edition. Now, evaluating low sperm concentrations (<2 × 10^6/mL) requires more precision, acknowledging that errors in counting small numbers of spermatozoa can be significant. Ensuring that examined aliquots represent the entire ejaculate and conducting enough observations to reduce random variability is essential.

To achieve acceptable performance, thorough in-house training is necessary. Basic medical laboratory standards require regular internal quality control to monitor interand intra-personal variability. Each laboratory should also participate in an external quality assessment (EQA) scheme. Proper interpretation of semen examination results depends on reference limits.

In the 6th Edition, sperm vitality assessment is recommended when total sperm motility is below 40%. Notably, terms like "normozoospermia," "asthenozoospermia," "necrozoospermia," and "teratozoospermia" are not used in this edition. These terms have been removed because reference thresholds alone are meaningless; multiple criteria must be applied to diagnose male infertility. While this approach is correct, clinicians may find the absence of reference values confusing. They may need to rely on other literature sources, which can be time-consuming and challenging. Consequently, clinicians might continue using the 5th centile values from the 5th Edition, designed to compare fertile and infertile men with a criterion of time to pregnancy ≤12 months.

Advanced Examination:

The 6th Edition introduces the SDF assay as an extended semen assessment that can be requested in specific clinical scenarios, although it does not provide guidance on testing indications or address test result variability across different SDF assays. The manual offers a detailed outline of the technical aspects of these tests and some guidance on interpreting the results. However, it lacks discussion on the indications and clinical application of these test results.

The extensive data on human semen in the 6th Edition provide valuable insights into the global management of male infertility. Expanding our knowledge and understanding of different aspects of human semen will optimize the care of infertile men and improve the reproductive outcomes of infertile couples. However, the lack of reference values for basic and advanced semen examinations in this edition might limit its global utilization, leading clinicians to continue using the older 5th Edition for patient management.

Note: The above commentary has been lightly edited for better flow.

Take Home Message: Contributing author - Ashok Agarwal

The 6th Edition of the WHO manual on semen analysis introduces updated reference values, emphasizes the inadequacy of fixed thresholds for diagnosing male infertility, and includes new tests for sperm DNA fragmentation (SDF) and seminal oxidative stress (OS). It provides detailed methodologies, stresses quality control, and re-adopts progressive motility distinctions. Obsolete tests are removed, and subjective semen odor evaluation is added. Terms like "normozoospermia" are eliminated to encourage a comprehensive diagnostic approach. These changes are supposed to improve the accuracy and usefulness of semen analysis in assessing male infertility.

However, the 6th Edition leaves several unresolved questions: the lack of clear indications for new tests like SDF and OS, variability in SDF test results without specified cut-offs, and inadequate guidelines for integrating advanced tests into clinical practice. Additionally, the subjectivity of semen odor measurement complicates standardization, and removing terms like "normozoospermia" could confuse clinicians.