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.

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.

Serum amyloid P component: a new biomarker for low sperm concentration?

Serum amyloid P component (SAP) is present in seminal plasma, on spermatozoa, and in different tissues of the male reproductive tract, but its function is not known. The aims of this study were to determine if the concentration of SAP in seminal plasma is associated with commonly assessed semen parameters and to investigate if SAP could be a new, indirect biomarker for these parameters. In a cross-sectional study of 203 young volunteers, the concentration of SAP in seminal plasma was measured with a in-house developed enzyme-linked immunosorbent assay. Scatter plots, Pearson's correlation coefficients (r), and linear regression models were produced, and SAP showed a statistically significant correlation with sperm concentration (r = 0.75), sperm number (r = 0.68), semen volume (r = -0.19), progressive sperm motility (r = 0.24), and sperm immotility (r = -0.20). When the study group was dichotomized, SAP could be used to discriminate samples with a sperm concentration < or ≥5 × 106 ml-1, 15 × 106 ml-1, or 40 × 106 ml-1, and in receiver operating characteristic curves, the corresponding areas under the curves were 0.97, 0.93, and 0.82, respectively, with P < 0.001 for all three cutoff values studied. The concentration of SAP in seminal plasma showed a strong, positive correlation with the concentration of spermatozoa in semen. SAP may be used as a new indirect potential biomarker for sperm concentration in fresh and in frozen, stored samples. In addition, it is envisaged that the assay could be developed into a home fertility test to differentiate between a low and a normal sperm concentration.

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.

Indication for Y Chromosome Microdeletion Analysis in Infertile Men: Is a New Sperm Concentration Threshold Needed?

OBJECTIVE: To describe the prevalence of Y-chromosome deletions in patients with a sperm concentration of less than 5 million/mL. To also determine a new sperm threshold for Y-chromosome analysis in men with infertility. METHODS: A total of 3023 patients who had a semen concentration of less than 5 million/mL included in this retrospective study. All of these patients had a genetic evaluation, hormonal evaluation, and 2 abnormal semen analyses. RESULTS: Y-chromosome deletions were present in 116 (3.8 %) patients with sperm concentration <5 million/mL. The frequency of a Y-chromosome deletions was 6.8%, 1.0%, 0.15% in azoospermic men, in men with sperm concentrations of 0-1 million /mL, in men with sperm concentrations of 1-5 million/mL. Patients were divided into 2 groups regarding the determined new sperm threshold. The sensitivity and specificity of the Y-chromosome deletions test were 92.2.7% and 49.3 %, 99.1%, and 22.1% in patients with azoospermia and sperm concentrations <1 million/mL, respectively. If the sperm concentration thresholds of azoospermia or <1 million/mL, are applied, the number of tests decreased to 50.5% (1442 tests) and 23.1% (643 tests), respectively. Approximately $108,150 and $48,225 would be saved if the sperm thresholds were azoospermia and <1 million/mL, respectively CONCLUSION: The current threshold of sperm concentration for Y-chromosome deletions is controversial. The new proposed sperm threshold for genetic testing of 1 million/mL would increase sensitivity and more cost-effective compared to the current threshold.

Microfabrication of low-cost customisable counting chambers for standardised estimation of sperm concentration.

Evaluation of sperm concentration is essential for research and procedures involving AI, cryopreservation and sperm quality assessment. Microfabrication technologies have shown tremendous potential for rapid prototyping and fabrication of devices to assist reproduction and fertility research, but such utility has not yet been made available for most reproduction laboratories. The aim of this study was to evaluate the feasibility of using microfabrication techniques to produce counting chambers for estimation of sperm concentration. Zebrafish (Danio rerio) spermatozoa were used as a model for evaluation of functionality of the chambers. These microfabricated enumeration grid chambers (MEGC) were composed of a polydimethylsiloxane (PDMS) coverslip with grid patterns (100 µm×100 µm) and a PDMS base platform to create a known volume with a 10-µm height to restrict the cells to a single layer. The results of cell counts estimated by two of three prototype MEGC devices tested were not significantly different from the control device, a commercially available Makler chamber. The material cost for a MEGC was less than US$0.10 compared with product costs of approximately US$100 for a standard haemocytometer and US$700 for a Makler counting chamber. This study demonstrates the feasibility of microfabrication in creating low-cost counting chambers to enhance standardisation and strengthen interdisciplinary collaborations.

Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen.

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml-1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

Hyaluronan bound mature sperm count (HB-MaSC) is a more informative indicator of fertility than conventional sperm parameters: Correlations with Body Mass Index (BMI).

The relationship between overweight and male fertility is well studied, still the correlation of obesity and decreased sperm quality is a subject to debate. The widely used conventional spermatological examinations alone seem to be inadequate to assess fertilization potential. Hyaluronan Binding Assay (HBA®) is one of the available validated tests that allows the functional examination of sperm. Data of 72 male patients (mean age 33.9 (24-43) years) from infertile couples were analysed. Body Mass Index (BMI) determination, conventional semen analysis and HBA were performed. Additionally, a relatively new Hyaluronan Bound Matured Sperm Count (HB-MaSC) -index, first introduced by the authors in 2015, was calculated. This index reflects fertilization potential of sperm more precisely. With the increase of BMI, sperm count decreased significantly until about 25 kg/m2, above 25 kg/m2 no further decrease was observed, although sperm count remained permanently low. Greater body weight (in the 70-90 kg range) was observed to have a significant negative effect only on the progressive sperm motility. In addition to sperm concentration and motility, sperm fertilization potential is also negatively affected by obesity, but is irrespective of body weight, as evaluated using BMI + HB-MaSC linear regression analyses adjusted for age and weight. This correlation between male BMI and sperm fertilization potential - as opposed to the conventional correlations with sperm concentration or motility - appears to provide more helpful information in the identification of real capability for fertilization.

Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen / 亚洲男科学杂志(英文版)

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml-1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

Novel centrifugal technology for measuring sperm concentration in the home.

OBJECTIVE: To evaluate the analytical performance and usability of the Trak Male Fertility Testing System, a semiquantitative (categorical) device recently US Food and Drug Administration (FDA)-cleared for measuring sperm concentration in the home by untrained users. DESIGN: A three-site clinical trial comparing self-reported lay user results versus reference results obtained by computer-aided semen analysis (CASA). SETTING: Simulated home use environments at fertility centers and urologist offices. PATIENT(S): A total of 239 untrained users. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm concentration results reported from self-testing lay users and laboratory reference method by CASA were evaluated semiquantitatively against the device's clinical cutoffs of 15 M/mL (current World Health Organization cutoff) and 55 M/mL (associated with faster time to pregnancy). Additional reported metrics include assay linearity, precision, limit of detection, and ease-of-use ratings from lay users. RESULT(S): Lay users achieved an accuracy (versus the reference) of 93.3% (95% confidence interval [CI] 84.1%-97.4%) for results categorized as ≤15 M/mL, 82.4% (95% CI 73.3%-88.9%) for results categorized as 15-55 M/mL, and 95.5% (95% CI 88.9%-98.2%) for results categorized as >55 M/mL. When measured quantitatively, Trak results had a strong linear correlation with CASA measurements (r = 0.99). The precision and limit of detection studies show that the device has adequate reproducibility and detection range for home use. Subjects generally rated the device as easy to use. CONCLUSION(S): The Trak System is an accurate tool for semiquantitatively measuring sperm concentration in the home. The system may enable screening and longitudinal assessment of sperm concentration at home. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02475395.

Establishing reference values for age-related spermatogonial quantity in prepubertal human testes: a systematic review and meta-analysis.

OBJECTIVE: To collect published data on spermatogonial quantity in the testes of healthy children and calculate the reference values of spermatogonial quantities throughout prepuberty. DESIGN: Systematic literature search in PubMed and EMBASE focusing on the number of spermatogonia per transverse tubular cross section (S/T) and spermatogonial density per cubic centimeter (cm3) of testicular volume (S/V) throughout prepuberty. SETTING: None. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Polynomial meta-regression analyses of S/T and S/V of healthy boys from the ages of 0 to 14 years. RESULT(S): We found six papers describing original quantitative data on S/T and S/V of healthy boys (total n = 334 and 62, respectively) that were suitable for meta-analysis. Polynomial meta-regression analyses of S/T and S/V demonstrated a clear pattern of spermatogonial quantity throughout prepubertal life. This consisted of a decline during the first 3 years of life, a gradual increase until the ages of 6 to 7 years, a plateau until the age of 11 years, and a sharp incline reaching pubertal numbers at 13 to 14 years of age. The association between S/T and S/V allowed us to perform S/T to S/V extrapolation, creating reference S/V (rS/V) values throughout prepubertal life from a cohort of 372 boys. CONCLUSION(S): Spermatogonial quantity varies during testicular development toward puberty. The values found in this study may serve as a baseline clinical reference to study the impact of diseases and adverse effects of gonadotoxic treatments on spermatogonial quantity in prepubertal testes. Spermatogonial quantity reference values may also help to evaluate the quality of testicular biopsy samples acquired for fertility preservation of prepubertal boys.

Effect of varicocele on semen characteristics according to the new 2010 World Health Organization criteria: a systematic review and meta-analysis.

This study investigated the effects of varicocele on semen parameters in infertile men based on the new 2010 World Health Organization laboratory manual for the examination of human semen. Semen analysis results (volume, sperm count, motility, and morphology) were the primary outcomes. An electronic search to collect the data was conducted using the Medline/PubMed, SJU discover, and Google Scholar databases. We searched articles published from 2010 to August 2015, i.e., after the publication of the 2010 WHO manual. We included only those studies that reported the actual semen parameters of adult infertile men diagnosed with clinical varicocele and contained a control group of either fertile men or normozoospermic men who were not diagnosed with varicocele. Ten studies were included in the meta-analysis, involving 1232 men. Varicocele was associated with reduced sperm count (mean difference: -44.48 × 10 [6] ml-1 ; 95% CI: -61.45, -27.51 × 10 [6] ml-1 ; P < 0.001), motility (mean difference: -26.67%; 95% CI: -34.27, -19.08; P < 0.001), and morphology (mean difference: -19.68%; 95% CI: -29.28, -10.07; P < 0.001) but not semen volume (mean difference: -0.23 ml; 95% CI: -0.64, 0.17). Subgroup analyses indicated that the magnitude of effect was influenced by control subtype but not WHO laboratory manual edition used for semen assessment. We conclude that varicocele is a significant risk factor that negatively affects semen quality, but the observed pooled effect size on semen parameters does not seem to be affected by the WHO laboratory manual edition. Given most of the studies published after 2010 still utilized the 1999 manual for semen analysis, further research is required to fully understand the clinical implication of the 2010 WHO laboratory manual on the association between varicocele and semen parameters.

Interferometric phase microscopy for label-free morphological evaluation of sperm cells.

OBJECTIVE: To compare label-free interferometric phase microscopy (IPM) to label-free and label-based bright-field microscopy (BFM) in evaluating sperm cell morphology. This comparison helps in evaluating the potential of IPM for clinical sperm analysis without staining. DESIGN: Comparison of imaging modalities. SETTING: University laboratory. PATIENT(S): Sperm samples were obtained from healthy sperm donors. INTERVENTION(S): We evaluated 350 sperm cells, using portable IPM and BFM, according to World Health Organization (WHO) criteria. The parameters evaluated were length and width of the sperm head and midpiece; size and width of the acrosome; head, midpiece, and tail configuration; and general normality of the cell. MAIN OUTCOME MEASURE(S): Continuous variables were compared using the Student's t test. Categorical variables were compared with the χ(2) test of independence. Sensitivity and specificity of IPM and label-free BFM were calculated and compared with label-based BFM. RESULT(S): No statistical differences were found between IPM and label-based BFM in the WHO criteria. In contrast, IPM measurements of head and midpiece width and acrosome area were different from those of label-free BFM. Sensitivity and specificity of IPM were higher than those of label-free BFM for the WHO criteria. CONCLUSION(S): Label-free IPM can identify sperm cell abnormalities, with an excellent correlation with label-based BFM, and with higher accuracy compared with label-free BFM. Further prospective clinical trials are required to enable IPM as part of clinical sperm selection procedures.

Accu-Beads as a quality control measure for manual and automated methods of measuring sperm concentration-an observational study.

PURPOSE: To determine the precision and accuracy of Accu-Beads and their utility as a quality control product for manual and automated measurements of sperm concentration. METHODS: This observational study was performed at an Assisted Reproductive Technology laboratory in a tertiary-care, university hospital. To simulate sperm concentration, bead concentrations were measured with the use of a manual and an automated method. RESULTS: The manual counts did not vary significantly from the automated counts regardless of the concentration. However, the counts did vary between lots of low concentration of Accu-Beads and between the two different types of fixed counting chambers. The two bead concentrations used in this study were below the 95 % confidence interval for the values listed by the manufacturer. CONCLUSION(S): In our laboratory, Accu-Beads met enough of the requirements of a good control material to be acceptable for daily quality control use, especially if we set our own ranges of acceptability for each vial of Accu-Beads. It is necessary to evaluate each new lot of Accu-Beads when they are received and again if they are used with a different counting chamber.

Methods for sperm concentration determination.

Proper assessment of the number of spermatozoa is essential not only as an initial step in every clinical infertility investigation [Björndahl et al (2010) A practical guide to basic laboratory andrology, 1st edn. Cambridge University Press, Cambridge] but also when attempting to establish the total sperm production in the testis [Amann (Hum Reprod 25:22-28, 2010); Amann (J Androl 30:626-641, 2009); Amann and Chapman (J Androl 30:642-649, 2009)]. Reliable methods combined with an understanding of the specific physiology involved as well as the main sources of errors related to the assessment of sperm concentration are critical for ensuring accurate concentration determination [Björndahl et al (2010) A practical guide to basic laboratory andrology, 1st edn. Cambridge University Press, Cambridge; World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen. WHO, Geneva]. This chapter therefore focuses on these three aspects.

Efecto de la cámara de recuento utilizada sobre los parámetros espermáticos, analizados con el ISASv1® / Effect of counting chamber on seminal parameters, analyzing with the ISASv1®

Introducción: El uso de las cámaras de recuento condiciona los valores de concentración y movilidad espermática debido a su tipo de carga (capilaridad o desplazamiento de la gota) y a la altura que presenten. Recientemente, se han introducido nuevas cámaras, tanto desechables (ISAS®D4C) como reusables (Spermtrack®) con diversas alturas en cada caso. Objetivo: El objetivo del presente trabajo es validar su uso, analizando diversos parámetros espermáticos con un sistema CASA, el ISASv1®. Material y método: Se analizaron muestras de donantes voluntarios (10 para las desechables y 5 para las reusables), analizando el efecto del tipo de cámara, de la altura, del tiempo de carga y del tiempo de análisis. Resultados: Los resultados obtenidos no mostraron diferencias significativas con el tiempo de carga para las cámaras reusables ni con el tiempo de análisis en ninguna de las cámaras. Discusión: La cámara reusable de 10 μm y las desechables de 10, 16 y 20 μm no mostraron diferencias entre sí, mientras que la reusable de 20 μm presentó resultados significativamente diferentes con respecto a las demás, por lo que su uso (siguiendo la recomendación de la Organización Mundial de la Salud) debe hacerse teniendo en cuenta este hecho. Conclusión: Las nuevas cámaras ofrecen un resultado muy homogéneo, con independencia del tiempo de carga o de análisis (AU)

Introduction: The use of counting chambers conditions the sperm concentration and motility values by their type of filling (capillary or drop displacement) and chamber height. We have recently introduced new chambers, both disposable (ISAS®D4C) and reusable (Spermtrack®), with different heights in each case. Objective: This study has aimed to validate its use by analyzing different sperm parameters with a CASA system, that is, the ISASv1®. Material and method: Samples from volunteer donors (10 for the disposable and 5 for reusable), analyzing the effect of chamber type, height, filling and analysis time, were used. Results: The results obtained showed no differences within the filling time for the reusable chambers, or the analysis time in any cases. Discussion: The 10 microns reusable chamber and disposable, 10, 16 and 20 microns showed no differences between them. However, the reusable 20 microns showed significantly different results, so its use (as recommended by WHO) should be taking this into account. Conclusion: The new chambers offer very consistent results, independent of filling or time of analysis (AU)