The World Health Organization (WHO) laboratory manual for the examination and processing of human semen provides internationally recognised guidelines that standardise how semen samples are analysed and interpreted. The WHO 6th Edition is the most up-to-date version and serves as a key reference for fertility clinics and andrologists when assessing male reproductive potential. The criteria used to interpret a semen analysis are based on several parameters, including semen volume, sperm concentration, total sperm number per ejaculate, sperm motility, and sperm morphology. This edition is based on data from 3,589 fertile men whose partners achieved pregnancy within 12 months, allowing results to be interpreted in relation to a well-defined reference population.
Compared with the WHO 5th Edition, the 6th Edition introduces several important refinements while maintaining a similar overall structure and reference distributions. Greater emphasis is placed on clinical relevance and methodological accuracy, with less reliance on strict reference limits alone. In basic semen analysis, the assessment of sperm numbers has been simplified through updated dilution methods, with a recommendation that at least 200 spermatozoa per replicate be counted to improve accuracy. Laboratories are also advised not to stop reporting sperm concentrations at low levels, such as 2 million/mL as suggested in earlier editions, but instead to report lower concentrations while acknowledging the increased uncertainty associated with very low sperm counts.
Sperm motility assessment in the 6th Edition has reverted to the traditional four-grade classification system: fast progressively motile, slow progressively motile, non-progressively motile, and immotile (grades a, b, c, and d). This change reflects the clinical importance of identifying rapidly progressive spermatozoa, as the presence of fast progressively motile sperm has been suggested to be more strongly associated with a higher likelihood of natural conception. For sperm morphology, the manual provides a detailed, systematic assessment approach, supported by improved high-quality micrographs of normal, borderline, and abnormal spermatozoa from unprocessed semen samples. These visual aids aim to improve consistency and training among laboratory personnel, particularly when evaluating subtle morphological features.
Normal results in standard semen parameters do not always reflect true functional fertility, as significant underlying abnormalities affecting sperm function may still be present. Extended examinations are therefore essential for identifying factors that are not detected through routine semen analysis alone, including the presence of leukocytes and immature germ cells, biochemical markers of accessory gland function, sperm aneuploidy, and genetic integrity. In particular, sperm DNA fragmentation has emerged as a key marker of male fertility, as increased DNA damage is associated with reduced chances of natural conception, impaired embryo development, and poorer reproductive outcomes, even when conventional semen parameters appear normal. Another important extended assessment is the mixed antiglobulin reaction (MAR) test, which evaluates the presence of antisperm antibodies bound to motile spermatozoa; these antibodies can impair sperm motility, disrupt interaction with cervical mucus, and inhibit sperm binding or penetration of the oocyte, thereby reducing fertility potential despite otherwise normal semen analysis results.
Although these WHO guidelines are essential for understanding male fertility health and ensuring consistency in semen analysis, it is important to recognise their limitations. Semen parameters should always be interpreted in the context of the individual patient and the couple as a whole. Values below the reference ranges do not necessarily mean that pregnancy cannot be achieved, just as results within the normal range do not guarantee pregnancy. Semen analysis results should always be interpreted alongside the patient’s medical history, physical examination, and factors relating to the female partner.
