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.

Comparative computer-assisted sperm analysis in non-human primates.

BACKGROUND: Biomedical research has recently focused on developing new models of human disease by implementing genome-editing strategies in non-human primates (NHPs) to introduce relevant gene mutations. There is a need to establish objective semen evaluation methods to select sires for in vitro fertilization to perform germline editing in embryos. METHODS: Sperm motility kinematic parameters were evaluated using a computer-assisted semen analysis (CASA) instrument for rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), and common marmosets (Callithrix jacchus). RESULTS: Normative sperm kinematic parameters were established, revealing differences between marmosets and macaques. The impact of season on rhesus macaque sperm motility was modest, where changes in sperm motility related to season were dependent on the individual male. CONCLUSIONS: These data provide a baseline of normative kinematic parameters for three captive NHP species, in which implementation of CASA may serve as a tool to evaluate NHP semen quality.

Performance of four chambers to measure sperm concentration: results from an external quality assurance programme.

RESEARCH QUESTION: What are the changes in the use of four types of counting chambers by laboratories enrolled in an Australian-based external quality assurance programme, and what are their accuracy and precision? DESIGN: Samples of latex beads of known concentration up to 20 × 106/ml were distributed quarterly to enrolled laboratories over a 12-year period. The results of each distribution were then used to calculate a bias relative to the target value as an indicator of accuracy and a coefficient of variation to indicate the level of precision. RESULTS: The proportion of laboratories in 2007-2008 using improved Neubauer haemocytometers (44%), Makler® (9%) and Vetriplast chambers (19%) remained constant in 2018-2019, unlike Kova chamber users (20%), which reduced. The mean (range) bias of improved Neubauer haemocytometers (-2.8% [-22.5 to +32.0%]) was less than Makler® chambers (+17.0% [-2.9 to +41.2%]), Kova chambers (+33.9% [0.0 to 115.0%]) and Vetriplast chambers (+47.9% [0.0 to 170.0%]). The coefficient of variation of improved Neubauer haemocytometers (14.6% [8.7 to 25.0%]) was less than both Vetriplast (20.7% [8.8 to 36.4%]) and Makler® (24.1% [13.6 to 48.6%]) and Kova chambers (35.5% [15.9 to 123.0%]). CONCLUSIONS: The improved Neubauer haemocytometer has been shown to be superior in accuracy and precision to the Makler®, Kova and Vetriplast chambers in their estimation of concentrations up to 20 × 106/ml. Users of Makler® chambers, specifically designed for counting spermatozoa, should take care to monitor the performance of their own chambers, whereas Kova and Vetriplast chambers (designed for microscopic urinalysis) should not be used.

At-home sperm testing for epidemiologic studies: Evaluation of the Trak male fertility testing system in an internet-based preconception cohort.

BACKGROUND: Semen quality assessment in population-based epidemiologic studies presents logistical and financial challenges due to reliance on centralised laboratory semen analysis. The Trak Male Fertility Testing System is an FDA-cleared and validated at-home test for sperm concentration and semen volume, with a research use only sperm motility test. Here we evaluate the Trak System's overall utility among men participating in Pregnancy Study Online (PRESTO), a web-based study of North American couples planning pregnancy. METHODS: US male participants aged ≥21 years with ≤6 months of pregnancy attempt time at study enrolment were invited to participate in the semen testing substudy after completing their baseline questionnaire. Consenting participants received a Trak Engine (battery-powered centrifuge) and two test kits. Participants shared their test results via smartphone images uploaded to online questionnaires. Data were then linked with covariate data from the baseline questionnaire. RESULTS: Of the 688 men invited to participate, 373 (54%) provided consent and 271 (73%) completed at least one semen test result. The distributions of semen volume, sperm concentration, motile sperm concentration, total sperm count, and total motile sperm count were similar to 2010 World Health Organization (WHO) semen parameter data of men in the general population. The overall usability score for the Trak System was 1.4 on a 5-point Likert scale (1 = Very Easy, 5 = Difficult), and 92% of participants believed they performed the test correctly and received an accurate result. Lastly, men with higher motile sperm count were more likely to report feeling "at ease" or "excited" following testing, while men with low motile sperm count were more likely to report feeling "concerned" or "frustrated." Overall, 91% of men reported they would like to test again. CONCLUSIONS: The Trak System provides a simple and potentially cost-effective means of measuring important semen parameters and may be useful in population-based epidemiologic fertility studies.

Evaluation of porcine semen quality by portable and desktop CASA systems - Short communication.

When using artificial insemination in porcine reproduction, one of the most important requirements is the suitable quality of semen regarding its total motility (TM) and progressive motility (PM). Computer-assisted sperm analysis (CASA) is an appropriate method to analyse the quality of semen. Recently a portable instrument has been developed to help specialists in their everyday field work. In our study, semen quality was measured simultaneously by the portable device (Ongo) and a laboratory CASA system (Microptic) to compare TM and PM values obtained by these appliances at a concentration of 50 × 106 spermatozoa/mL. Agreement between measurements was evaluated with a Bland-Altman plot. Strong correlation was found between the investigated instruments for all the three parameters, i.e. sperm concentration, TM and PM. However, a few measurements fell outside the defined range of acceptance.

Automation of human semen analysis using a novel artificial intelligence optical microscopic technology.

Current semen analysis still commonly depends on a manual microscopy method in clinical laboratories worldwide. However, some of the major disadvantages of this technique are that it is labour-intensive, subjective, laboratory-based and time-consuming. Although computer-assisted semen analysers (CASAs) have enabled partial automation of routine semen analysis, they lack wider acceptance due to their complicated operation. Therefore, the development of an accessible, rapid and standardised method for semen analysis is urgently needed. Here, we describe the development and clinical testing of a novel, automated, artificial intelligence optical microscopic (AIOM)-based technology, LensHooke™ X1 PRO (X1 PRO), designed for the quantitative measurement of sperm concentration, motility and seminal pH. We observed high degree of correlation in the results of concentration, progressive motility and progressively motile sperm concentration between the X1 PRO semen analyser and manual method using 135 clinical semen samples. In addition, the seminal pH results obtained by X1 PRO and manual methods were comparable (p = .12). In summary, our results showed that new X1 PRO semen analyser is a reliable diagnostic tool for routine semen analysis providing clinically acceptable results based on World Health Organization (WHO) 5th Edition guidelines.

Computer-aided sperm analysis, the new key player in routine sperm assessment.

For sperm analysis, important inter-laboratory variations have been observed in manual analyses. In this study, a computer-aided sperm analysis (CASA) system was assessed versus manual technique, and specific software modifications were operated to fit the David's classification already used in the laboratory. Four parameters were studied (concentration, motility, vitality and morphology), and at least 30 semen samples from 30 different patients have been tested. Manual and automated analyses were compared using a least-squares regression line analysis, Student's t test, Bland-Altman plots and Passing-Bablok regressions. Repeatability was also assessed, and coefficients of variation (CV) were calculated. Both manual and automated methods gave similar results for sperm concentration (n = 150), motility (n = 30), vitality (n = 90) and morphology (n = 90). Repeatability always showed a decrease in the CV with automated analysis; for example in normal range of sperm values, CV for manual and CASA analyses were, respectively, 9.0% versus 4.4% for sperm concentration, 5.2% versus 4.1% for motility, 7.3% versus 4.2% for vitality and 11.4% versus 4.1% for morphology. All parameters were comparable between automated and manual analysis, and repeatability measures confirm the more reliable values of the SCA compared to those of manual analysis.

Combined effect of type and capture area of counting chamber and diluent on Holstein bull sperm kinematics.

The evaluation of sperm motion is crucial for processing of seminal doses for artificial insemination. Here, the combined effect of the type and capture area of three counting chambers, together with the type of diluent employed, on sperm motility was analysed. Ejaculates from thirteen Holstein bulls were used for sperm kinematic analysis with the ISAS® v1 CASA-Mot system, using two capillary-loaded counting chambers (Leja® and Cell-Vu® ) and one drop displacement chamber (Makler® ). Nine fixed positions were analysed per chamber type, considering central and lateral and three longitudinal fields. Independent of the diluent used, differences were found between the three chambers. Independent of the extender, no differences in x-axis were observed with Cell-Vu® , while using Leja® , some parameters showed lower values in the centre than in lateral areas. In both counting chambers, the lowest values were observed in the distal area. Results obtained with the two diluents were highly different with a very low correlation between them. In conclusion, the capture area inside the chambers leads to significant changes in sperm kinematic parameters and different dilution media introduce considerable differences in the motility patterns. It is necessary to optimise sampling methods and specific set-ups to be used with CASA-Mot technology.

Automated semen analysis by SQA Vision® versus the manual approach-A prospective double-blind study.

Next to clinical investigations, the evaluation of male fertility relies mainly on detailed sperm analyses, for example, cell counting, motility, cell morphology and vitality testing. The manual creation of a spermiogram is time- and material-consuming. Therefore, reliable high-throughput systems that may be substituted for manual methods are urgently needed. The present study aimed to compare conventional sperm analysis performed as per WHO 5th guidelines and semen analysis performed with the SQA Vision® machine. SQA Vision® is a commercial device for automated sperm analysis. Data obtained independently by both methods were compared by statistical analyses using Bland-Altman plots and Passing-Bablok regression analyses. The analyses revealed that the results for sperm concentration and total motility were comparable. The agreement for progressive motility was poor, and there were clear deviations in the determination of normal sperm morphology. Passing-Bablok regression analyses and the consideration of the 95% confidence intervals pointed out systematic and proportional differences between the manual semen analysis and the automated approach.

Method agreement between three different chambers for comparative boar semen computer-assisted sperm analysis.

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 106  sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 µm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non-progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.

Validation of a portable device (iSperm® ) for the assessment of stallion sperm motility and concentration.

The objective of this study was to evaluate the accuracy of a novel, portable device (iSperm® Equine for assessing concentration and motility of stallion semen). In the first experiment, semen concentration was determined by the iSperm® Equine (Aidmics Biotechnology), Androvision® (Minitube) and NucleoCounter® SP-100™ (ChemoMetec). The total motility and progressive motility were determined by the iSperm® Equine and the Androvision® using the manufacturer's guidelines. Frozen/thawed semen samples (n = 33) at various dilutions were analysed for concentration and motility with the above-mentioned devices. There was a significant correlation between the concentrations measured with iSperm® and NucleoCounter® at all the measured dilutions. Moreover, <10% difference in concentrations was observed between the iSperm® and NucleoCounter® using the Bland-Altman test. There was also a significant correlation between iSperm® and Androvision® for total and progressive motility. In the second experiment, the parameters used in the Androvision® were modified to match those of the iSperm® . Total motility and progressive motility of frozen/thawed semen samples (n = 10) were determined, and the similarity between the Androvision® and iSperm® was confirmed by correlation studies and Bland-Altman test. The results of these experiments demonstrate that the iSperm® offers a reliable and practical alternative for the semi-automated measurement of concentration and motility of stallion semen in the field. The iSperm® enables the practitioner to obtain objective and repeatable measurements on a variety of semen types (fresh, cooled and frozen) in the field at the time of insemination and thus acquire more insight into the quantity and quality of the provided insemination doses. This mare-side diagnostic tool may help practitioners in identifying presumed subfertility problems more rapidly and act accordingly.

Evaluation of a portable device for assessment of motility in stallion semen.

In horse breeding, quality assessment of semen before insemination is often requested. Non-laboratory-based techniques for objective analysis of sperm motility are thus of interest. The aim of this study was evaluating a portable device for semen analysis (Ongo sperm test) and its comparison with computer-assisted semen analysis (CASA). Semen was collected from 10 stallions, diluted to 100, 50 and 25 × 106  sperm/ml and analysed for total (TM) and progressive motility (PM). The final sperm concentration influenced total motility analysed by Ongo (p < 0.05) which was higher at 100 × 106  sperm/ml when compared to 25 × 106  sperm/ml (p < 0.05) but not when compared to 50 × 106  sperm/ml (n.s.). Sperm concentration did not influence total motility when assessed by SpermVision (n.s.). Agreement between methods was evaluated by correlation analysis and Bland-Altman plot. Intra-assay variation of Ongo was 5.2% ± 3.0 for TM and 6.9% ± 3.4 for PM. Correlation between Ongo and CASA was r = 0.79, 0.88 and 0.83 for 100, 50 and 25 × 106  sperm/ml for TM, and r = 0.87, 0.89 and 0.87 for PM, respectively (all p < 0.001). At the 100 and 25 mio/ml dilutions, the difference between the two systems deviated significantly from 0, while no such bias existed at the 50 mio/ml dilution (TM Ongo 85.0%, CASA 82.3%; PM Ongo 64.1%, CASA 66.1%). The 95% confidence interval was 19.9%, 18.9% and 19.2% ± mean for TM and 20.7%, 17.4% and 20.3% ± mean for 100, 50 and 25 × 106  sperm/ml, respectively. In conclusion, Ongo sperm test sperm motility data were strongly correlated with data obtained by CASA. In addition, at a concentration of 50 × 106  sperm/ml values measured with both systems were close to identical. At this concentration, which is recommended in equine AI, Ongo and CASA can be used interchangeably.

[IVOS â ¡ versus Sperm Class Analyzer in the results of semen analysis].

OBJECTIVE: To compare the results obtained from the computer-aided sperm analysis (CASA) systems of the two fully-automated commercial sperm quality analyzers, Hamilton-Thorn IVOS Ⅱ (IVOS Ⅱ) and Spanish Sperm Class Analyzer (SCA). METHODS: A total of 99 semen samples were collected in the Center of Reproduction of Shenzhen Zhongshan Urology Hospital from September 2018 to October 2018 and, according to the sperm concentration, divided into groups A (<15 ×106/ml), B (15－50 ×106/ml) and C (>50 ×106/ml). IVOS Ⅱ, SCA and manual microscopy were used for the examination of each sample, followed by comparison of the sperm concentration, sperm motility and percentage of progressively motile sperm (PMS) obtained from IVOS Ⅱ and SCA. RESULTS: The sperm concentrations derived from IVOS Ⅱ and SCA were significantly higher than that from manual microscopy in group A (ï¼»10.24 ± 4.60ï¼½ and ï¼»10.20 ± 5.11ï¼½ vs ï¼»8.45 ± 4.15ï¼½ ×106/ml, P < 0.05), but showed no statistically significant difference in group B (ï¼»30.95 ± 11.84ï¼½ and ï¼»31.81 ± 12.90ï¼½ vs ï¼»29.14 ± 10.65ï¼½ ×106/ml, P > 0.05) or C (ï¼»102.14 ± 45.97ï¼½ and ï¼»109.48 ± 46.32ï¼½ vs ï¼»104.74 ± 41.87ï¼½ ×106/ml, P > 0.05). Significant differences were not observed between IVOS Ⅱ and SCA in the percentage of PMS (ï¼»24.21 ± 14.62ï¼½% vs ï¼»23.92 ± 15.42ï¼½%, P > 0.05) or sperm motility (ï¼»37.48 ± 19.34ï¼½% vs ï¼»37.69 ± 16.61ï¼½%, P > 0.05) in group B, nor in group C (PMS: ï¼»30.80 ± 12.06ï¼½% vs ï¼»32.98 ± 16.10ï¼½%, P > 0.05; sperm motility: ï¼»44.50 ± 15.62ï¼½% vs ï¼»47.26 ± 17.46ï¼½%, P > 0.05). Both the percentage of PMS and sperm motility obtained from IVOS Ⅱ were remarkably lower than those derived from SCA in group A (PMS: ï¼»18.54 ± 12.96ï¼½% vs ï¼»22.90 ± 12.88ï¼½%, P < 0.05; sperm motility: ï¼»26.97 ± 14.05ï¼½% vs ï¼»34.90 ± 15.18ï¼½%, P < 0.05). IVOS Ⅱ and SCA both showed a high repeatability (CV <15%), and the former exhibited an even higher one than the latter, in detection of sperm concentration, sperm motility and the percentage of PMS. CONCLUSIONS: IVOS Ⅱ and SCA both had a good consistency in the results of sperm concentration, motility and progressive motility, but showed a poor comparability with low-concentration semen samples.

[Hamilton-Thorne Integrated Visual Optical System â (IVOSâ ) versus IVOS â ¡ for human sperm analysis].

OBJECTIVE: To compare the computer-assisted sperm analysis (CASA) systems Hamilton-Thorne Integrated Visual Optical System Ⅰ (IVOSⅠ) and IVOS Ⅱ after verifying the performance of the latter so as to ensure the accuracy of the results of analysis. METHODS: Based on the criteria established in the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen (WHO 5th Ed), we compared the main semen parameters obtained from IVOSⅠ with those generated by IVOS Ⅱ, and examined the consistency between the results of the two sperm analyzers. RESULTS: The linear relationship of the outlier test, bias estimation and scatter plot and the results of the outlier test of the two systems all met the requirements of comparison analysis and showed an obvious correlativity. The application scope of the results obtained from the apparatus indicated a reasonable value range, with r = 0.988 for sperm concentration, r = 0.975 for sperm progressive motility (PR), and r = 0.981 for total sperm motility. Evaluation of the acceptability of the predicted bias showed that the allowable total error (TEa) to be 6.67% with sperm concentration at 12 × 106 /ml and 2.34% with PR < 31%, their upper limit of the allowable error < 1/2. The results of IVOS Ⅱ conformed to the requirements of the WHO 5th Ed. CONCLUSIONS: The main parameters derived from IVOSⅠ and IVOS Ⅱ are comparable and consistent, indicating that both can be used for the examination of semen samples.

Microfluidic sorting selects sperm for clinical use with reduced DNA damage compared to density gradient centrifugation with swim-up in split semen samples.

STUDY QUESTION: Does microfluidic sorting improve the selection of sperm with lower DNA fragmentation over standard density-gradient centrifugation? SUMMARY ANSWER: Microfluidic sorting of unprocessed semen allows for the selection of clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation. WHAT IS KNOWN ALREADY: Microfluidic devices have been explored to sort motile and morphologically normal sperm from a raw sample without centrifugation; however, it is uncertain whether DNA damage is reduced in this process. STUDY DESIGN, SIZE, DURATION: This is a blinded, controlled laboratory study of differences in standard semen analysis parameters and the DNA fragmentation index (DFI) in split samples from infertile men (n = 70) that were discarded after routine semen analysis at an academic medical center. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sperm concentration, progressive motility and forward progression were assessed by microscopic examination. For each sample, the unprocessed semen was tested for DNA fragmentation and split for processing by density-gradient centrifugation with swim-up or sorting by a microfluidic chip. DNA fragmentation was assessed in unprocessed and processed samples by sperm chromatin dispersion assay. The DFI was calculated, from up to 300 cells per slide, as the number of cells with fragmented DNA divided by the number of cells counted per slide. MAIN RESULTS AND THE ROLE OF CHANCE: The median DFI in unprocessed samples was 21% (interquartile range (IQR): 14-30). In paired analyses of all samples, those processed by the microfluidic chip demonstrated significantly decreased DFI compared to those processed by density-gradient centrifugation (P = 0.0029) and unprocessed samples (P < 0.0001). The median DFI for chip specimens was 0% (IQR: 0-2.4) while those processed by density-gradient centrifugation had a median DFI of 6% (IQR: 2-11). Unprocessed samples in the highest DFI quartile (DFI range: 31-40%) had a median DFI of 15% (IQR: 11-19%) after density-gradient centrifugation and DFI of 0% (IQR: 0-1.9%) after processing with the microfluidic chip (P = 0.02). LIMITATIONS, REASONS FOR CAUTION: While a high DFI has been associated with poor outcomes with IVF/ICSI, there are limited data illustrating improvements in clinical outcomes with a reduction in DFI. As this study utilized discarded, non-clinical samples, clinical outcomes data are not available. WIDER IMPLICATIONS OF THE FINDINGS: While microfluidic sorting of unprocessed semen allowed for the selection of clinically usable, highly motile sperm with nearly undetectable levels of DNA fragmentation, standard processing by density-gradient centrifugation with swim-up did not increase DNA fragmentation in an infertile population. The proposed microfluidic technology offers a flow-free approach to sort sperm, requiring no peripheral equipment or filtration step, while minimizing hands-on time. STUDY FUNDING/COMPETING INTEREST(S): No external funding to declare. Utkan Demirci, PhD is the Co-founder and Scientific Advisor for DxNow Inc., LevitasBio Inc. and Koek Biotech. Mitchell Rosen, MD is a member of the Clinical Advisory Board for DxNow Inc.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

Quantitative assessment of heavy metal effects on sperm function using computer-aided sperm analysis and cytotoxicity assays.

One known environmental risk factor impacting on human reproduction is heavy metal pollution. Although some metals (e.g., Cu, Se and Zn) have protective effects on the male reproductive system in low doses, heavy metals can accumulate to toxic levels and result in poor semen quality and decreased sperm function. We investigated the effect of CuSO4 and CdCl2 (10, 50, 100 and 250 µg/ml or 500 µg/ml) on human sperm motility and vitality by using computer-aided sperm analysis (CASA) and two cytotoxicity assays (WST-1 and XTT). Several sperm motility parameters were significantly reduced after 5 hr of exposure to the highest concentrations of CuSO4 (250 µg/ml) and CdCl2 (500 µg/ml). The WST-1 assay also revealed significantly lower absorbance values for 50, 100 and 250 µg/ml CuSO4 and for 500 µg/ml CdCl2 ; however, no significant effect was seen with XTT. The calculated average IC50 value was 50.31±  4.34 µg/ml for CuSO4 and 392.32  ±76.79 µg/ml for CdCl2 . The effects of these metals were confirmed with MgCl2 , a positive control. This study provides threshold concentrations for the harmful effect of CuSO4 and CdCl2 on human spermatozoa and recommends the use of WST-1 as vitality assay in future in vitro studies.

Evaluation of the Sysmex UF-1000i system as an alternative for the screening of genital tract inflammation in male infertility patients.

The number of white blood cell (WBC) in semen is an important indicator of genital tract inflammation in male infertility. The peroxidase assay is the recommended reference method for seminal WBC counting. However, it is time-consuming and may cause relatively heavy workload in daily routine. Meanwhile, the main component in the reagent of peroxidase test is harmful to human and the environment. In this study, we evaluated the analytical performance of the Sysmex UF-1000i that is a urine flow cytometer as a screening tool for genital tract infection in male infertility patients through the counting of seminal WBC. We examined 143 semen samples and compared the results of UF-1000i and manual microscopy. The intra-assay variability, stability and linearity studies were performed. The intravariability (CV %) of seminal WBC count by Sysmex UF-1000i was 2.34%-9.65%. The method of UF-1000i displayed a good agreement with the reference assay of manual microscopy, and the r value for correlation of seminal WBC count between UF-1000i and manual microscopy was over 0.999 (p < 0.001). The Sysmex UF-1000i is capable of producing reliable seminal WBC count consistent with that obtained by manual microscopy. It is a suitable alternative to the manual microscopy, thus reduces the workload.

Subjective and objective assessment of fish sperm motility: when the technique and technicians matter.

Fish sperm motility is nowadays considered the best sperm quality biomarker in fish, and can be evaluated both by subjective and computerized methods. With the aim to compare the precision and accuracy of both techniques, fish sperm samples were assessed by subjective methods and by a computer-assisted sperm analysis (CASA-Mot) system, and simultaneously by three different technicians with different degrees of expertise on the sperm quality analysis. Statistical dispersion parameters (CV, coefficient of variation; and RG, range) were estimated in order to determine the precision and accuracy of the techniques and the influence of laboratory staff on sperm motion assessments. Concerning precision, there were not much significant differences between the technical support staff (high, medium, and low experimented technician), and statistical dispersion parameters were quite similar between them independent of the technique used and the sperm motility class analyzed. However, concerning accuracy, experimented technician reported subjective motility values very closed to the values provided by the CASA-Mot system, only 10 percentage points away from the data provided by a CASA-Mot system. However, medium and low experimented technicians often overestimate the CASA-Mot values, and amplitudes up to 30 percentage points were detected in several sperm assessments. To sum up, both the technique (subjective or objective) and the technician (degree of expertise) became key factors in order to reach accurate motility estimations, so the use of both qualified staff and novel CASA-Mot systems seems to be a critical requirement for obtaining satisfying results in fish species with similar motility patterns.

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTION: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (P < 0.05) in the choline/glycerophosphocholine (GPC), lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40%/80% interface. WHAT IS KNOWN ALREADY: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Sperm suspended in phosphate buffered saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80%' sperm. MAIN RESULTS AND THE ROLE OF CHANCE: DGC with two washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination while retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/GPC 1H MRS peak greater than 3:1 signal to noise ratio (SNR) was estimated at ~3 × 106/ml. The choline/GPC and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (P < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. LIMITATIONS, REASONS FOR CAUTION: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. WIDER IMPLICATIONS OF THE FINDINGS: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest.