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Asian Journal of Andrology logoLink to Asian Journal of Andrology
. 2024 Jan 16;26(3):229–232. doi: 10.4103/aja202380

What is required for better progress in clinical and scientific andrology involving sperm assessments?

Lars Björndahl 1,
PMCID: PMC11156447  PMID: 38265240

In scientific publications based on basic semen examination, there is a general lack of consistency and transparency regarding laboratory techniques used. To support improvements in clinical and scientific andrology, it is essential to increase understanding of basic physiology and biology, and enhance compliance with existing recommendations and international standards. Study designs need to be improved as well as transparency of laboratory methods used and open publication of data obtained.

Semen examination is still the cornerstone of investigations of male factors of infertility.1 Many attempts have been tried to establish a direct relation between different semen characteristics and couple infertility, but with no or very limited success. It is likely that this perceived lack of usefulness is due to different misapprehensions. The first problem is the difficulty to isolate male factors contributing significantly to couple infertility. If all female infertility factors were known and well defined, it would be possible to define male factors better. Assisted reproductive technologies evolved into useful clinical methods for couples to achieve pregnancies even though the man may have signs of disturbed fertility potential.2 However, this has for a long time overridden the use of semen examination as a tool to understand the functions of the male reproductive organs.

Recent findings indicate that abnormal of semen examination results is linked to later morbidity and shorter life expectancy.3,4,5 This further underlines that semen examination is important as an investigation of the man, and that the man, not only his sperm, is the patient.

Still, it must be emphasized that the basic semen examination cannot answer all questions. More research in this field is warranted, which requires that the basic semen examination provides reliable results. A recent study6 of 122 articles in Human Reproduction and Fertility and Sterility between 2011 and 2020 revealed that true compliance with World Health Organization (WHO) recommendations was not possible to understand from the vast majority of these publications. It was common to cite WHO recommendations, but not report any details of the actual techniques used. It should be noted that this study did not include reports using computer-aided sperm analysis system (CASA) for any of the measured characteristics in the ejaculates since CASA is not recommended by the WHO for examination of ejaculates.7

The aim of this article is to elucidate aspects important to consider when designing better studies as well as improving the interpretation of semen examination results in research and in clinical investigations. The purpose is also to inspire investigations of neglected aspects of human male fertility functions, and improvement of existing techniques and assessments.

Increased awareness of the basic physiology and biology, understanding of limitations of present WHO reference limits, differences between in vivo and in vitro circumstances, importance of proper laboratory techniques, personnel training, quality control, study design, and publication of results is crucial for the advancement of clinical and scientific andrology.

UNDERSTANDING BASIC PHYSIOLOGY AND BIOLOGY

It appears to be a common belief that something called semen exists in the male body, and that the ejaculation produces a homogenous body fluid. This is, however, not the case. The ejaculate is formed during ejaculation, and the different ejaculate fractions are not equivalent.8 In the normal ejaculation, the first few fractions contain the majority of spermatozoa together with prostatic fluid, while the last approximately 2/3 of the ejaculate is dominated by seminal vesicular fluid.9,10 In vivo, it is most probable that the first ejaculated, rapidly progressive spermatozoa quickly move into the cervical mucus and start the passage to the site of fertilization.11 It is described that maybe 100–1000 spermatozoa in an ejaculate will ever be close to encounter the oocyte in the fallopian tube.12 These potentially successful spermatozoa are not likely to have had any significant contact with the seminal vesicular fluid.11,13 This is important since seminal vesicular fluid is known to reduce sperm motility and sperm survival.9,10,14 Furthermore, exposure of spermatozoa to seminal vesicular fluid induces changes in the sperm chromatin structure. This can jeopardize the delivery of a complete and undamaged genome (DNA) into the oocyte.15 Nevertheless, it must not be forgotten that assessment of number of spermatozoa in the ejaculate can give valuable information about the man’s production and transport of spermatozoa. Furthermore, detailed sperm morphology assessment at the light microscope level can give important explanations of unsuccessful fertilization, for instance, reduced sperm motility (gross tail abnormalities or disturbance of mitochondrial sheath in mid piece of sperm). An example that semen examination may reveal specific problems in a man is that in some men with ejaculatory duct obstruction (EDO), spermatozoa can exhibit poor sperm motility in spite of a good sperm number.16 It can be speculated that with EDO, most sperm will not be expelled with prostatic fluid. Instead, spermatozoa will be pressed into the urethra first when the seminal vesicles have built up a pressure and started to empty. The direct contact between spermatozoa and seminal vesicular fluid would be detrimental for several sperm capacities.

LIMITATIONS OF PRESENT WORLD HEALTH ORGANIZATION REFERENCE LIMITS

The present recommendations by the WHO include suggestions regarding reference limits.7,17 Such limits are certainly important for the interpretation of semen analysis results, but it has been put in question that the suggested reference limits should represent limits between fully fertile men and men with reduced potential for fertility.2,7,18 From a biological point of view, one could expect that there are many different causes for reduced male fertility potential. It would then not be probable that all known or unknown causes should have the same reference limits.

Another problem is how the reference limits have been established. The basis was both 2010 and 2021 results from men in couples who have succeeded in starting a pregnancy within 12 months of trying. This is based on the WHO definition of couple infertility and is also a practical time frame for when investigation and treatment of the couple should be started. However, the 12-month “time-to-pregnancy” does not automatically reflect the absence of male infertility factors. Already 70 years ago, it was clear that, in couples succeeding to start a pregnancy early, the men had higher results for sperm concentration and motility than men in couples who had to try for longer time.19 This was also observed in an independent study in 1968, showing that most couples started a pregnancy within 3–4 months.20 In a recent study, it was also shown that men, in couples starting a pregnancy within a few months, on average had semen examination results much higher than the present reference limits suggested by the WHO.21 Still, the reference limits presented in 202117 give guidance by showing whether a man’s semen examination results are among those of <5% of men in the mixed reference population who had started a pregnancy within 12 months.

It is very important that the WHO reference limits are not used as limits between fertility and infertility when defining the fertility status of an individual man or a study group.

DIFFERENCE BETWEEN CIRCUMSTANCES IN VIVO AND IN VITRO

A main difference between the in vivo situations compared to the circumstances commonly in vitro is that in vivo exposure of spermatozoa to seminal vesicular fluid is minimal.9,10,11,14 Another difference is that after in vitro handling of spermatozoa, apparently higher numbers of spermatozoa must be present around the oocyte(s) to ascertain that fertilization will occur. This indicates that most spermatozoa handled with usual routines may not be able to fertilize an oocyte. If this is true, it means that the routine handling of ejaculates and spermatozoa does not provide a reliable source of information related to sperm capacity to fertilize oocytes. It also means that, in general, in vitro selection of spermatozoa appears much less efficient and specific than the natural selection by the passage through the cervical mucus and further transporting it to the site of fertilization.

LABORATORY TECHNIQUES

Basic semen examination is well described both in the WHO manual7 and in the International Standard Organization (ISO) standard.22 The essential base for the techniques is to ensure reliable results – minimizing technical variations by, for instance, using adequate equipment (pipettes and microscopes), investigating representative aliquots (sufficient aliquot volume and comparing replicate assessments), maintaining controlled temperature for motility assessments, using proper staining for morphology and vitality assessments, implementing proper training of laboratory personnel, performing internal quality control (IQC), and participating in extern quality assessments.

If other equipment or procedures instead of those described in the standard are to be used, the laboratory must validate that the equipment or procedures will still provide results that are fully comparable to the recommended ones. If new, but elsewhere validated, equipment is to be used, the laboratory still must verify that the results obtained with the new equipment are in compliance with the standards.

PERSONNEL TRAINING

Since so much of the basic semen examination relies on microscopic assessments, the training of the personnel is essential.23,24 There are courses that provide the theory and basic practical training, but for an individual to perform with good accuracy and acceptable variability, further training is required.25,26,27,28,29 When the basic training is finished, it is still necessary to perform regular IQC to ensure low inter- and intra-individual variability.7,22

QUALITY CONTROL

The International Standard for Medical Laboratories (ISO 15189:2022)30,31 as well as the Specific Standard for Basic Semen Examination (ISO 23162:2021)22 specify, like the WHO manual,7 that IQC and external quality assessment (EQA) must be implemented.

Compared to common biochemical laboratory control, laboratory andrology is based on microscopic observations. Thus, there are no standard substances to order from a chemical company. A more complex material must be produced in order to measure the capability of laboratory personnel to make correct observations. On the other hand, in comparison with machines, well-trained laboratory personnel are able to supervise their work, not the least with the comparison of replicate assessments. Therefore, the daily testing, as required when machines are involved in producing quantitative data, is superfluous in microscopic work. Still, the IQC and the EQA must be performed regularly to detect any drift in assessments. The frequency varies with the number of ejaculates examined in the laboratory. It may be practical to calculate the number of annual IQC samples by the total number of samples examined. If a total of 1000 clinical examinations are done in a year, it can be reasonable to do 10 IQC samples in a year. However, in a laboratory performing only 100 yearly examinations, still a monthly IQC is reasonable, and also if each individual performs very few examinations. It should also be noted that, when vasectomy controls and controls of suspected cases of azoospermia or severe oligozoospermia are performed, the skill to recognize immotile or motile spermatozoa is established within IQC of motility and counting of spermatozoa in fertility examinations.

Both in IQC and EQA, it is important that the test material resembles the assessments performed in routine work. It can be a practical problem if several members of personnel shall assess motility on a fresh sample since assessments must be done almost simultaneously to avoid differences due to the biological deterioration of motility of spermatozoa stored in vitro. This is also a problem in EQA, where recorded video clips are the only practical possibility. Still, it is essential that video recordings and routine assessments are as similar as possible, concerning magnification, brightness, and contrast. One way to enhance the similarity between routine and IQC/EQA is to perform motility assessments on the same screen as the IQC/EQA of recorded video clips. For sperm counting, IQC can more easily be performed on the same day by all personnel. For EQA, only sperm suspensions where bacterial overgrowth is reduced by an additive can be used. To reduce health hazards if formaldehyde (formalin) is used as a fixative, the concentration should be much lower than what is used in, for instance, pathology laboratories fixing organ and tissue samples. IQC of sperm morphology can be done on stained and mounted slides. For EQA, stained and mounted slides can be used for assessment, and only fixed slides for evaluating local staining as well as assessment. The ability to differentiate between dead and live spermatozoa can be assessed in IQC on fresh sample as long as preparation of slides is done reasonably simultaneously (confer motility). Otherwise, the same methods as those for EQA should be used. For both IQC and EQA, assessment of stained and permanently mounted slides can be used.

IQC

The IQC has several levels. The basic level is the standard operating procedures. The use of sufficient aliquot volumes and number of assessed spermatozoa is required to reduce error due to random poor representativity of the examined aliquot. The mandatory comparison of repeat assessments is also required to reduce risk for poor representativity of the specimen examined.

To control that there is no systematic drift in results over time, regularly recurrent examination of material by all personnel performing analyses in the laboratory is mandatory. Such supervision is also necessary to establish that analysis results are not varying substantially between different analyzers. How IQC assessments can be evaluated is described in detail and step-by-step in the WHO manual.7

EQA

The purpose of EQA is primarily to ascertain that the assessments of the laboratory are comparable to other laboratories. In the best-case scenario, the EQA scheme bases the target values on assessments from well-trained personnel in laboratories with a record of long-time experience in laboratory andrology. If the EQA scheme only reports variability and mean values of all participating laboratories, there is a risk that both mean varies and variability measures are too “lenient”, allowing substantial errors in the evaluation of results.

EQA scheme may distribute material with different regularities. It appears that two annual distributions are the most common. For international schemes, it appears that it can be difficult to distribute material more than twice yearly due to different seasons between the northern and southern hemispheres as well as considerable national variations in the allocation of traditional annual holidays.

STUDY DESIGN

For the design of a useful and valid study, the study groups (including control groups) must be properly defined.32,33,34 Therefore, male fertility potential cannot be based on the WHO reference limits for semen examination. Furthermore, how well the female partners are examined is essential. All semen examinations should be done according to standards, IQC performed, and the participation of a qualified, necessary EQA scheme.

Besides basic semen examination results, it is necessary that the study includes data of importance for routine clinical investigations. This includes the base for positive predictive values (PPVs) and negative predictive values (NPVs). Clinically important tests cannot only rely on correlations or P-values for differences between groups. Correlations are sufficient to indicate possible relations or differences between study group and control group and for conclusions on the group level. However, due to usual variability in tests, the usefulness of a test in a clinical setting, when conclusions shall be drawn for an individual patient or couple, requires the establishment of PPV and NPV. Furthermore, it is important to relate PPV and NPV to the frequency of the disorder in the examined population. With different prevalence of a disorder, the PPV and NPV will vary.

Another important aspect is the choice of proper outcome measures. Comparing a new test with basic semen examination results has a very limited usefulness. A high degree of correlation with results from basic semen examination clearly indicates that the test does not contribute to new information. Any important outcome measure that does not correlate with basic semen examination can be useful if itself has a reasonably low variability, is fairly easy to perform, and is not too expensive in equipment or labor cost.

PUBLICATION OF RESULTS

Within the field of biological research, there are now increasing demands for improvements of scientific reporting.33,35,36 The background is a general lack of reproducibility of published results. New demands require both substantially better and more transparent statements of study populations and laboratory investigations performed.32,33,34 There are also requirements that data underlying published studies should be openly available for the scientific community, provided that confidential information of the research participants is not disclosed.33

A vast majority of studies concerning male fertility factors that have been published in leading scientific journals do not appear to bother to present the laboratory methods used to obtain the data.6 This is a severe problem since the use of data obtained with suboptimal quality procedures and methods is associated with high technical variability. This is likely to conceal true correlations and differences.

There is a special responsibility of scientific journals to ascertain that published studies are based on reliable laboratory techniques and that publications are transparent regarding the techniques to allow independent repeats of the studies.34,37 To support the work of editors and reviewers, tools in the form of checklists have been developed,32,33,34 but not until very recently implemented as compulsory in manuscript submission and made available to the readers as supporting information.37

CONCLUSIONS

To support the progress in clinical and scientific andrology, a number of aspects concerning the study design and performance, and control of basic semen examination, require several improvements. This is also pertinent to the publication of results from such studies.

COMPETING INTERESTS

The author declares no competing interests.

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