Effect of the technical variability of counting chambers upon the interpretation of sperm concentration results.

RESEARCH QUESTION: Does the choice of sperm-counting chamber affect the proportion of samples generating results with an erroneous interpretation? DESIGN: Laboratories in an external quality assurance programme were sent 141 semen samples over a 12-year period and asked to return the sperm concentration and whether or not the result was abnormal. Only those using 5th edition of the World Health Organization manual (WHO5) interpretation criteria were included. Submissions from specialist fertility laboratories were used to calculate assigned values for each sample. Laboratory50 values determined the sperm concentration at which the laboratories reported a majority transition from abnormal to normal interpretations, i.e. the tipping point, which should coincide with the lower reference limit. RESULTS: The median and range of bias from the assigned values of each sample were determined for the Makler (-3.3%; -88.6% to +332.8%), haemocytometer (10.6%; -93.3% to +645.5%), Kova (+65.3%; -71.7% to +581.8%) and Vetriplast (+72.4%; -100.0% to +709.1) chambers. Laboratory50 values for the Makler (17.3  ×  106/ml), haemocytometer (13.6  ×  106/ml), Kova (10.0  ×  106/ml) and Vetriplast chambers (8.8  ×  106/ml) reflected the under- and overestimation of the chambers and confirmed a shift in the adjusted lower reference limit then used. The proportion of laboratories reporting erroneous interpretations of the four chambers for oligozoospermic samples were 10.9%, 15.1.%, 40.1% and 44.0%, respectively, and rose as the adjusted lower reference limit decreased. CONCLUSIONS: The between-laboratory and within-sample variation for all the chambers was high and remains a concern. The main impact of an increasing bias of the chambers was a lowering of the laboratory50 tipping point, resulting in an under-reporting of abnormal semen samples.

Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

OBJECTIVE: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. METHODS: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. RESULTS: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. CONCLUSIONS: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Extended semen examinations in the sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen: contributing to the understanding of the function of the male reproductive system.

In the sixth edition of the World Health Organization manual for the examination and processing of human semen, extended examination methods to provide key diagnostics in the investigation of the male reproductive system function are elaborated. These go beyond the basic analysis of semen and may be useful in more specifically guiding the clinical characterization of fertile or infertile men. Among the extended examinations included in the chapter, the use of multiparametric scoring for sperm morphological defects, sperm DNA fragmentation, and the roles for computer-assisted analysis of sperm or semen are arguably those that will be the most widely used and may also cause the most debate.

The past, present, and future of the semen analysis.

This month's Views and Reviews provides an added perspective to the World Health Organization laboratory manual for the examination and processing of human semen, which was recently published in the 6th edition. The first artice provides a historical context of the prior editions of the World Health Organization manuals and modifications adopted over the years. The next piece then provides additional perspectives on the methodologies used for the performance of semen analysis. The third article then examines some of the new semen analytic technologies and enhancements that have become more common over the years. Finally, the last article proposed where male reproductive testing will head in the coming years with emerging research and technologies.

The sixth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen: ensuring quality and standardization in basic examination of human ejaculates.

A basic semen investigation has established principles that are necessary for ascertaining reliable and internationally comparable results. Although these principles have been present in the WHO manual since its inception, the baseline issue across most published studies and practice in reproductive medicine (in which the male is considered) is repetitive failure to adhere to these principles, thereby leading to relevant comparable data and accuracy. To address this failure, the sixth edition of the WHO manual includes revised basic methods, and a complementary formal standard of the International Standards Organization (ISO23162:2021) for basic semen examination has been published. Perhaps the most significant change in the sixth edition is the reintroduction of the four-category distinction of sperm motility, which causes additional work for laboratories in changing reporting parameters but is clinically important. Another essential change is the widened focus from mainly a prognostic tool for medically assisted reproduction to additionally raising awareness of semen examination as a measure of male reproductive functions and general male health.

Survey of semen analysis practices in India and need for standardization and improvement.

BACKGROUND AND OBJECTIVES: Infertility is a sensitive subject carrying with it economic, social, and psychological implications. Work up of male infertility is often hampered by a lack of infrastructure and facilities, as well as inadequate training of pathology residents. The purpose of this research survey was to evaluate the current status of semen analysis practices and compare them to the standards laid down by the World Health Organization (WHO). MATERIALS AND METHODS: A web-based questionnaire was designed consisting of questions related to semen analysis practices and procedures being followed currently by pathologists in India. A total of 194 pathologists responded. Questions regarding the procedures followed for semen collection, sperm count, volume, normal range, lower normal limit of sperm count, morphology, etc., were included in the survey. These data were recorded. The differences and gaps in the practice with respect to the WHO standards were analyzed. RESULTS: The survey revealed that the printed instructions for semen analysis were available with 38.7% of the respondents; 58.8% of the respondents had a separate room for semen collection; 95.9% performed the analysis manually, and; only 4.1% used automated analyzers. Only 53.6 and 52.6% of the respondents were correctly reporting the normal range of semen volume and sperm counts, respectively. Only 19.6% stated as having read the WHO manual and were also practicing its guidelines, while 14.4% had not read the WHO manual even once. CONCLUSION: The present study showed a large gap between the practice of semen analysis by respondents from various parts of India and standard procedures as laid down by the WHO. Many laboratories do not follow the standard instructions. There is a need to improve the quality of practice related to semen analysis in this country through appropriate teaching and training in medical institutions as well as through Continuing Medical Education (CMEs) or regular update programs.

Evolution of the WHO "Semen" processing manual from the first (1980) to the sixth edition (2021).

As stated clearly in all editions of the WHO Laboratory Manual for the Examination and Processing of Human Semen, the goal of the manual is to meet the growing needs for the standardization of semen analysis procedures. With constant advances in andrology and reproductive medicine and the advent of sophisticated assisted reproductive technologies for the treatment of infertility, the manual has been continuously updated to meet the need for new, evidence-based, validated tests to not only measure semen and sperm variables but also to provide a functional assessment of spermatozoa. The sixth edition of the WHO manual, launched in 2021, can be freely downloaded from the WHO website, with the hope of gaining wide acceptance and utilization as the essential source of the latest, evidence-based information for laboratory procedures required for the assessment of male reproductive function and health.

Comparison of two automated sperm analyzers using 2 different detection methods versus manual semen assessment.

PURPOSE: The exploration of male infertility is mainly based on semen analysis, but its evaluation might be affected by the operator's competence and subjectivity. This led to the development of automated semen analyzing systems. Despite continuous improvement, the precision and correlation of these automated systems with manual sperm assessment performed strictly according to WHO guidelines remains variable in the literature, and their role in daily practice is debated. METHODS: In this double blind prospective study, we compared the results provided by 2 automated systems based on different concepts (CASA and electro-optical signal) with manual sperm assessment. Sperm concentration, motility and morphology were performed simultaneously and independently by different operators, blinded to each other. RESULTS: A total of 102 unselected men attending the andrology department for routine sperm analysis were included in the study. We found no significant difference between each automated method and manual assessment for all sperm parameters, except for sperm morphology assessment where the electro-optical system gave higher results and performed slightly poorer than CASA. Correlation was moderate to high between manual assessment and each automated methods for all sperm parameters, with randomly distributed differences. CONCLUSIONS: Overall, these results show that both types of automated systems can be implemented in andrology laboratory for routine sperm analysis.

Abnormal fertilization in ICSI and its association with abnormal semen parameters: a retrospective observational study on 1855 cases.

Intracytoplasmic sperm injection (ICSI) efficiently addresses male factor infertility. However, the occurrence of abnormal fertilization, mainly characterized by abnormal pronuclei (PN) patterns, merits investigation. To investigate abnormal fertilization patterns following ICSI and identify their respective associations with abnormal parameters in semen analysis (SA), a retrospective observational study including 1855 cycles was performed. Male infertility diagnosis relied on the 2010 WHO criteria. The population was divided into groups based on their SA results. The presence of 2PNs and extrusion of the second polar body (PB) indicated normal fertilization. A Kruskal-Wallis test along with a Wilcoxon post hoc evaluation and Bonferroni correction was employed for comparison among the groups. For the pregnancy rate, logistic regression was employed. No correlation was established between the SA abnormalities and the 1PN or 3PN formation rates. The highest and lowest 0PN rates were reported for the oligoasthenoteratozoospermic and normal groups, respectively. The lowest cleavage formation rates were identified in the oligoasthenozoospermic and oligoasthenoteratozoospermic groups. The aforementioned groups along with the oligoteratozoospermic group similarly presented the lowest blastocyst formation rates. For the clinical pregnancy rate, no statistically significant difference was observed. In conclusion, the incidence of two or more abnormal SA parameters - with the common denominator being oligozoospermia - may jeopardize normal fertilization, cleavage, and blastocyst rates. Once the developmental milestone of achieving blastocyst stage status was achieved, only oligoasthenozoospermia and oligoasthenoteratozoospermia were associated with lower rates. Interestingly, following adjustment for the number of blastocysts, no statistically significant differences were observed.

Development and validation of a novel mail-in semen analysis system and the correlation between one hour and delayed semen analysis testing.

OBJECTIVE: To develop and validate a novel, mail-in semen analysis (SA) system. DESIGN: Prospective cohort. SETTING: Not applicable. PATIENT(S): Ejaculates from normospermic men. INTERVENTION(S): One-hour SA, then repeat SAs (on same ejaculate) over 52 hours using a novel technique for maintaining sperm viability. MAIN OUTCOME MEASURE(S): World Health Organization SA parameters. RESULT(S): One-hour SA on 104 ejaculates in the validation phase of the study demonstrated normal semen parameters. With up to 52 hours of observation and four subsequent SA measurements/ejaculate, concentration remained stable, motility decreased by 0.39%/h, and normal morphology decreased by 0.1%/h. Measured 1-hour and calculated motility (correlation coefficients 0.87) and morphology (correlation coefficients 0.82) strongly were correlated. CONCLUSION: This novel, mail-in, Clinical Laboratory Improvement Amendments-approved SA testing system demonstrates a strong degree of correlation between 1-hour and delayed SA testing. Given the linear motility and morphology decrease and stability of sperm concentration, this test may be used in clinical practice to evaluate semen quality for fertility evaluations. Furthermore, this approach significantly improves the ease, comfort, and efficiency of obtaining a SA, likely breaking down early barriers to accessing successfully a male fertility evaluation.

Male infertility.

It is estimated that infertility affects 8-12% of couples globally, with a male factor being a primary or contributing cause in approximately 50% of couples. Causes of male subfertility vary highly, but can be related to congenital, acquired, or idiopathic factors that impair spermatogenesis. Many health conditions can affect male fertility, which underscores the need for a thorough evaluation of patients to identify treatable or reversible lifestyle factors or medical conditions. Although semen analysis remains the cornerstone for evaluating male infertility, advanced diagnostic tests to investigate sperm quality and function have been developed to improve diagnosis and management. The use of assisted reproductive techniques has also substantially improved the ability of couples with infertility to have biological children. This Seminar aims to provide a comprehensive overview of the assessment and management of men with infertility, along with current controversies and future endeavours.

[Internal quality control products for computer-assisted sperm analysis: Research and application].

OBJECTIVE: To investigate the application of the self-made semen quality control (QC) product in internal QC of computer-assisted sperm analysis (CASA). METHODS: CASA was calibrated with high- and low-concentration commercially available semen QC product and meanwhile 15 samples of self-made mixed semen QC product were placed in 75 cryotubes containing liquid nitrogen, followed by CASA of the concentration, motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR) of the sperm using standard procedures and 50 days of continuous monitoring. The Makler counting plate was used to measure the concentration and motility of the self-made sperm. RESULTS: The coefficients of variation (CV) of the commercially available semen QC product at high and low concentrations were 6.18% and 7.85%, respectively. CASA showed that the concentration of the self-made QC product was (25.97 ± 1.41) ×106/ml, with a CV of 5.42%, and the sperm motility, VCL, VSL, VAP, LIN, WOB and STR were (22.15 ± 1.75)% (CV = 7.9%), (59.18 ± 2.05) µm/s (CV = 3.46%), (26.79 ± 1.2) µm/s (CV = 4.48%), (34.98 ± 1.4) µm/s (CV = 4.01%), 46.81 ± 1.55 (CV = 3.3%), 60.52 ± 1.3 (CV = 2.15%) and 76.46 ± 1.98 (CV = 2.59%), respectively. The concentration and motility of the self-made sperm detected with the Makler counting plate were (34.39 ± 2.37) ×106/ml (CV = 6.89%) and (38.04 ± 1.69)% (CV = 4.44%), respectively. Levey-Jennings QC charts were plotted for the indicators using the means and standard deviation. CONCLUSIONS: The self-made internal QC product by liquid nitrogen cryopreservation is feasible and effective for monitoring the accuracy and precision of CASA-derived sperm concentration and motion parameters, and it has a smaller CV than the commercially available QC product in measuring sperm concentration.

Discrepancy between Post-Vasectomy Semen Analysis Recommendation and Practice Patterns in the Post-2012 American Urological Association Guideline Era.

PURPOSE: In 2012 the American Urological Association published vasectomy guidelines to promote best practices, including when to obtain post-vasectomy semen analyses. In this study we assessed practice patterns of post-vasectomy semen analysis since this guideline publication. MATERIALS AND METHODS: We retrospectively analyzed a database of men who underwent post-vasectomy semen analysis between 2013 and 2017. Vasectomies were performed by urologist and nonurologist providers in academic and community settings. RESULTS: A total of 4,827 men underwent post-vasectomy semen analysis with 22.3% undergoing 1 or more repeat analyses. On initial analysis 58.2% were azoospermic, 28.3% had less than 100,000/ml rare nonmotile sperm, 8.7% had greater than 100,000/ml nonmotile sperm and 4.8% had motile sperm. The rate of repeat post-vasectomy semen analysis decreased from 30.7% in 2013 to 18.6% in 2016. Overall 72% of repeat post-vasectomy semen analyses were performed for patients with azoospermia or rare nonmotile sperm on initial post-vasectomy semen analysis. Of the 421 men with greater than 100,000/ml nonmotile sperm, 61.3% did not obtain a repeat analysis. Among cases of repeat analysis after initially having greater than 100,000/ml nonmotile sperm, 67.5% were downgraded to rare nonmotile sperm or azoospermia, 32.5% had a persistent count greater than 100,000/ml nonmotile sperm and none developed motile sperm. CONCLUSIONS: The rate of repeat post-vasectomy semen analysis is decreasing, likely highlighting a decrease in unnecessary testing. However, there is ongoing discordance between vasectomy guidelines and practice patterns, with 72% of repeat post-vasectomy semen analyses obtained unnecessarily based on guideline recommendations. Interestingly, no men with greater than 100,000/ml nonmotile sperm went on to have motile sperm on repeat post-vasectomy semen analysis. Further provider education is warranted and subsequent studies may allow for guideline modification wherein all nonmotile sperm are characterized similarly.

SARS-CoV-2 infection, male fertility and sperm cryopreservation: a position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) (Società Italiana di Andrologia e Medicina della Sessualità).

PURPOSE: The recent pandemic of severe acute respiratory syndrome (SARS) due to coronavirus (CoV) 2 (SARS-CoV-2) has raised several concerns in reproductive medicine. The aim of this review is to summarize available evidence providing an official position statement of the Italian Society of Andrology and Sexual Medicine (SIAMS) METHODS: A comprehensive Pubmed, Web of Science, Embase, Medline and Cochrane library search was performed. Due to the limited evidence and the lack of studies, it was not possible to formulate recommendations according to the Oxford 2011 Levels of Evidence criteria. RESULTS: Several molecular characteristics of the SARS-CoV-2 can justify the presence of virus within the testis and possible alterations of spermatogenesis and endocrine function. Orchitis has been reported as a possible complication of SARS-CoV infection, but similar findings have not been reported for SARS-CoV-2. Alternatively, the orchitis could be the result of a vasculitis as COVID-19 has been associated with abnormalities in coagulation and the segmental vascularization of the testis could account for an orchitis-like syndrome. Finally, available data do not support the presence of SARS-CoV-2 in plasma seminal fluid of infected subjects. CONCLUSION: Data derived from other SARS-CoV infections suggest that in patients recovered from COVID-19, especially for those in reproductive age, andrological consultation and evaluation of gonadal function including semen analysis should be suggested. Studies in larger cohorts of currently infected subjects are warranted to confirm (or exclude) the presence of risks for male gametes that are destined either for cryopreservation in liquid nitrogen or for assisted reproduction techniques.

[Updates in protocol for human semen examination.]

Currently, the WHO laboratory manual for the examination and processing of human semen (5th edition, 2010) provides updated, standardized, evidence-based procedures and recommendations for laboratory managers, scientists and technicians to follow in examining human semen in a clinical or research setting. Despite the fact, there are several gaps and limitations in the interpretation of this compendium. Mostly, the WHO-protocol of estimation of peroxidase-positive cells and spermatozoa, as well as evaluation of their viability and morphology are not so affordable and applicable in Russia due to peculiarities of laboratory market. Furthermore, most of Russian manuscripts do not reflect a unified approach to the analytical stage of semen analyses. In order to standardize the protocol for human semen examination which adopted to Russian lab it was developed packing of reagents (GEMSTANDART-SEMEN ANALYSES, LLC 'GEMSTANDART', Saint Petersburg, Russia) allowing to obtain an accuracy and completeness of the examination. In summary, this approach is a necessary step in male fertility evaluation. Together with a clinical information, it is indispensable for planning the appropriate clinical management and tacking care in male health.

Is azoospermia the appropriate standard for post-vasectomy semen analysis? Or an unachievable goal of best practice laboratory guidelines.

The increasingly stringent laboratory-approach to diagnosing azoospermia for post-vasectomy semen analysis (PVSA) continues to be at odds with the simpler approach desired by clinicians. This study describes the analysis of 10 years of PVSA and discusses the outcome in relation to risk, cost and assesses whether more stringent procedures are required. PVSA was performed on 4788 patients initially using a 2-test strategy (16 and 20 weeks post-surgery), moving to 1 test during 2013-2014. Azoospermia was confirmed by the analysis of 10 µl of semen followed by 10 µl of centrifuged pellet. In total, there were 9260 tests with a median of 1.93 tests/patient and 18.7 weeks to clearance. Surgical failure occurred in 1.75%, falling to 1.1% between 2011 and 2016. There were no cases of unwanted pregnancy, recanalization or complaints although misdiagnosis was detected in 1 case as a result of failure to confirm patient identification. Azoospermia performed according to World Health Organization (WHO) guidelines is sufficiently robust to confirm success/failure of vasectomy. With uncertainty surrounding the diagnosis, efforts to improve detection of occasional non-motile sperm are futile, cost more and fail to reduce risk of inappropriate clearance. Misdiagnosis is more likely from patient identification error and mitigation may include reverting to the safety net of a 2-test strategy.

Internal quality control products for computer-assisted sperm analysis: Research and application / 中华男科学杂志

Objective@#To investigate the application of the self-made semen quality control (QC) product in internal QC of computer-assisted sperm analysis (CASA).@*METHODS@#CASA was calibrated with high- and low-concentration commercially available semen QC product and meanwhile 15 samples of self-made mixed semen QC product were placed in 75 cryotubes containing liquid nitrogen, followed by CASA of the concentration, motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR) of the sperm using standard procedures and 50 days of continuous monitoring. The Makler counting plate was used to measure the concentration and motility of the self-made sperm.@*RESULTS@#The coefficients of variation (CV) of the commercially available semen QC product at high and low concentrations were 6.18% and 7.85%, respectively. CASA showed that the concentration of the self-made QC product was (25.97 ± 1.41) ×10⁶/ml, with a CV of 5.42%, and the sperm motility, VCL, VSL, VAP, LIN, WOB and STR were (22.15 ± 1.75)% (CV = 7.9%), (59.18 ± 2.05) μm/s (CV = 3.46%), (26.79 ± 1.2) μm/s (CV = 4.48%), (34.98 ± 1.4) μm/s (CV = 4.01%), 46.81 ± 1.55 (CV = 3.3%), 60.52 ± 1.3 (CV = 2.15%) and 76.46 ± 1.98 (CV = 2.59%), respectively. The concentration and motility of the self-made sperm detected with the Makler counting plate were (34.39 ± 2.37) ×10⁶/ml (CV = 6.89%) and (38.04 ± 1.69)% (CV = 4.44%), respectively. Levey-Jennings QC charts were plotted for the indicators using the means and standard deviation.@*CONCLUSIONS@#The self-made internal QC product by liquid nitrogen cryopreservation is feasible and effective for monitoring the accuracy and precision of CASA-derived sperm concentration and motion parameters, and it has a smaller CV than the commercially available QC product in measuring sperm concentration.

Semen and reproductive hormone parameters in fertile men with and without varicocele.

Although varicoceles are a widely accepted identifiable male factor in infertile couples, the benefit of varicocele repair in improving pregnancy and live birth rates remains uncertain. The Study for Future Families obtained semen and reproductive hormone samples from US men whose partners were currently pregnant. In our analysis cohort of 709 men, a varicocele was detected by clinical examination in 56 (8%) of men. Men with varicocele had smaller left testis, and lower total and total motile sperm counts than men without varicocele. Gonadotropin levels were higher as well in men with varicocele. Interestingly, testosterone levels were also slightly higher in men with varicocele. Despite these differences, there was no difference between the groups in the time to achieve the study pregnancy or percentage of men with a previous pregnancy. We conclude that even in fertile men, varicoceles are associated with some degree of testicular hypofunction. This would support current recommendations to consider varicocele repair in male partners in infertile couples who demonstrate both a varicocele and abnormal semen parameters and after evaluation for treatable female factors.

Effect of low sperm quality on progeny: a study on zebrafish as model species.

Nowadays a decrease tendency in human sperm quality has been reported mainly in developed countries. Reproductive technologies have been very valuable in achieving successful pregnancies with low quality sperm samples. However, considering that spermatozoa molecular contribution is increasingly important in recent studies, it is crucial to study whether fertilization with low sperm quality could leave a molecular mark on progeny. This study explores the consequences that fertilization with low sperm quality may have on progeny, using zebrafish as a model. Good and bad breeders were established attending to sperm quality analyses and were individually tracked. Significant differences in fertilization and malformation rates were obtained in progenies between high and low quality sperm samples. Moreover an altered miR profile was found in the progenies of bad zebrafish breeders (upregulation of miR-141 and miR -122 in 24 hpf embryos) and as a consequence, some of their targets involved in male sex development such as dmrt1, suffered downregulation. Our results indicate that fertilizing with high sperm quality samples becomes relevant from a new perspective: to avoid molecular alterations in the progeny that could remain masked and therefore produce unexpected consequences in it.