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.

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.

The influence of the female reproductive tract and sperm features on the design of microfluidic sperm-sorting devices.

Although medical advancements have successfully helped a lot of couples with their infertility by assisted reproductive technologies (ART), sperm selection, a crucial stage in ART, has remained challenging. Therefore, we aimed to investigate novel sperm separation methods, specifically microfluidic systems, as they do sperm selection based on sperm and/or the female reproductive tract (FRT) features without inflicting any damage to the selected sperm during the process. In this review, after an exhaustive studying of FRT features, which can implement by microfluidics devices, the focus was centered on sperm selection and investigation devices. During this study, we tried not to only point to the deficiencies of these systems, but to put forth suggestions for their improvement as well.

Buchholzia coriacea (wonderful kola) seeds induce male reproductive toxicity by suppressing the pituitary-gonadal axis in Wistar rats

The methanolic extract of Buchholzia coriacea seeds (MEBC) has been reported to induce male reproductive toxicity by decreasing sperm parameters and fertility index. To elucidate the possible mechanism(s), the effects of graded doses of MEBC on sex hormones and sperm profile were investigated in this study. The MEBC (e.g., 50, 200, 400, and 600 mg/kg) was administered daily (p.o.) to male Wistar rats for 6 weeks, while a concurrent control group received distilled water (vehicle). Then, the animals were sacrificed under sodium pentobarbital anaesthesia. Weights of organs were recorded, and the sperm profile was determined microscopically. Testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) were assayed from the obtained serum using the ELISA technique. Sperm motility was significantly reduced by MEBC (i.e., 50 and 200 mg/kg), and sperm count reduced in all treated groups in a dose-dependent manner compared with that of the control. Serum testosterone, LH, and FSH decreased in treated rats. A histopathological examination of testes showed a considerable depletion and necrosis of the epithelium of seminiferous tubules. The result suggests that Buchholzia coriacea seeds induce male reproductive toxicity by suppressing the pituitary-gonadal axis.

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.

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.

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.

Combined effects of type and depth of counting chamber, and rate of image frame capture, on bull sperm motility and kinematics.

Semen quality assessment requires accurate, reliable and objective methods for examination of sperm variables including sperm motility. For preparation of semen samples for artificial insemination, as a genetic resource, samples that are used for insemination need to have the capacity to result in a highly acceptable fertility rate. Several methods have been developed for evaluation of bull sperm in laboratory conditions and for preparation of doses for artificial insemination. Computer-assisted semen analyses can provide objective information on various sperm variables. Nevertheless, this equipment requires fine-calibrations considering differences among species, breeds and conditions for sample evaluation and data analyses. In the present study, there was examination of the interaction between factors such as image frame rate and type and depth of counting chamber in which sperm were evaluated, together with differences between bulls of four breeds. The use of the Spermtrack® reusable 10 µm-depth chamber provided more reliable results than results obtained using disposable chambers (10 and 20 µm depth). A capture rate of at least 90 fps is required for assessment of sperm motility percentage, whereas a rate of 250 fps is needed for obtaining consistent kinematic data. Differences among breeds in the present study indicate conditions for sperm analyses should include specific equipment calibrations for each breed. These results contribute to development of more precise conditions for assessments of bull sperm quality taking into account breed differences and the requirement each breed has for the adequate evaluation and preparation of samples for artificial insemination.

Fertility Enhancing Potency of Omega-3 Fatty Acids in Male Rats

Abstract Cases of some grave side effects of some allopathic medicines used in treatment of infertility has caused a global need for alternatives with minimal or no side effect, hence the demand to evaluate the fertility enhancing potential of omega-3 fatty acids (O3FA) in male rats. This study evaluated the fertility enhancing potential of omega-3 fatty acids (O3FA) in male rats. Seventy-two sexually mature male albino rats 11-13 weeks of age, weighing between 171 - 180 g were assigned into six groups (I - VI) fed graded doses of O3FA. Administration of O3FA lasted twenty-eight days at 48 hour intervals. At the end of the treatment, organosomatic index of testes, testicular and epididymis sperm cells counts and testicular histology were assessed following standard methods. The actual and relative testicular weights, testicular and epididymis sperm counts of all O3FA treated rats were significantly increased (p < 0.05) when compared with the control group rats. The photomicrographs of testes in O3FA treated rats showed normal spermatogonial cell layers and active spermatogenesis with appearance of spermatids in the lumen of some tubules. The findings of this study depicts that O3FA possesses the potency of enhancing various fertility indices in male rats with regards to absolute and relative testicular weights as well as sperm counts.

Home sperm testing device versus laboratory sperm quality analyzer: comparison of motile sperm concentration.

OBJECTIVE: To test the ability of the smartphone-based YO Home Sperm Test to accurately and precisely measure motile sperm concentration (MSC) versus the SQA-Vision, an automated laboratory semen analyzer. DESIGN: MSC compared for the YO device on Galaxy and iPhone smartphones versus the SQA-Vision in a double-blind manner. SETTING: Academic medical center. PATIENT(S): Donor semen samples from 24 men in 144 aliquots. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Accuracy, precision, and agreement assessed between the YO device and the SQA-Vision for MSC results. RESULT(S): The YO device demonstrated good correlation and good to moderate agreement with the SQA-Vision for MSC results up to a range of 94 × 106/mL with Pearson and concordance correlation coefficient above 0.92. The YO also showed a very high level of accuracy (97.8%) with positive and negative percent agreement above 94%. The difference in coefficient of variation between the YO and the SQA-Vision was low (between 9.4% and 11.2%) and not statistically significant. The precision among the YO phone devices was lower (16.0%) than the manufacturer's claim of ≤20%. CONCLUSION(S): The smartphone-based device has a high level of accuracy and precision when compared with the SQA-Vision. It can detect samples with abnormally "low" MSC (below 6 × 106/mL cutoff), which supports its use as an effective home sperm test for screening "low" and "moderate/normal" MSC cases. In addition, the device effectively identifies varying levels of normal MSC in a precise manner over a wide range of normal MSC. Thus, the YO Score can improve patient satisfaction and empowerment.

Effect of chamber characteristics, loading and analysis time on motility and kinetic variables analysed with the CASA-mot system in goat sperm.

Several factors unrelated to the semen samples could be influencing in the sperm motility analysis. The aim of the present research was to study the effect of four chambers with different characteristics, namely; slide-coverslip, Spermtrack, ISAS D4C10, and ISAS D4C20 on the sperm motility. The filling procedure (drop or capillarity) and analysis time (0, 120 and 240s), depth of chamber (10 or 20µm) and field on motility variables were analysed by use of the CASA-mot system in goat sperm. Use of the drop-filling chambers resulted in greater values than capillarity-filling chambers for all sperm motility and kinetic variables, except for LIN (64.5% compared with 56.3% of motility for drop- and capillarity-filling chambers respectively, P<0.05). There were no significant differences in total sperm motility between different chamber depths, however, use of the 20µm-chambers resulted in greater sperm progressive motility rate, VSL and LIN, and less VCL and VAP than chambers with a lesser depth. There was less sperm motility and lesser values for kinetic variables as time that elapsed increased between sample loading and sperm evaluation. For sperm motility, use of droplet-loaded chambers resulted in similar values of MOT in all microscopic fields, but sperm motility assessed in capillarity-loaded chambers was less in the central fields than in the outermost microscopic fields. For goats, it is recommended that sperm motility be analysed using the CASA-mot system with a drop-loaded chamber within 2min after filling the chamber.

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.

Aggravation of cyclophosphamide-induced reproductive toxicity in mice by aqueous extract of Aegle marmelos (L. )

ABSTRACT Aegle marmelos (L.) (Rutaceae) commonly known as bael is an important medicinal fruit tree. The present study focused on the effects of aqueous extract of Aegle marmelos (AEAM) on the testis and sperm characteristics induced by cyclophosphamide (CPA) in mice. Thirty six adult Parke's strain mice were divided into six groups: group I given only distilled water (control); group II administered with AEAM alone once in a week for five weeks; group III administered with CPA (200 mg/kg b.w., intraperitoneally) once in a week for five weeks and group IV-VI CPA along with AEAM (400, 500 and 600 mg/kg b.w., orally). CPA was found to reduce gonadosomatic index (GSI), sperm counts, motility, viability, antioxidant activities and induced histopathological changes of testis. In the group administered AEAM with CPA an exacerbation of sperm count, motility and viability of the cauda epididymis, GSI, antioxidant activities and architecture of testis was observed. The results suggest that the administration of AEAM may aggravate CPA-induced reproductive toxicity. It may be helpful in preparation of natural male contraceptives.

Andrology laboratory review: Evaluation of sperm concentration.

This article is the result of the work of the andrology task-force of the Association of Applied Animal Andrology, American College of Theriogenologists, European College of Animal Reproduction, Society for Theriogenology, and National Association of Animal Breeders. It is intended to serve as a comprehensive reference on methods to evaluate sperm concentration and to contribute to the adoption of best practices in veterinary andrology laboratories. The information covered in the article includes sample preparation and the use of manual counts, spectrophotometers, computer-assisted semen analysis, NucleoCounter, and flow cytometry. Emphasis is given to the principles of the methods and equipment, performing the evaluation, and common mistakes and/or pitfalls. In addition, the precision and accuracy of the different methods are also discussed.

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.

Influence of chamber type integrated with computer-assisted semen analysis (CASA) system on the results of boar semen evaluation.

The objective of the study was to evaluate the effect of different types of chambers used in computer-assisted semen analysis (CASA) on boar sperm concentration and motility parameters. CASA measurements were performed on 45 ejaculates by comparing three commonly used chambers: Leja chamber (LJ), Makler chamber (MK) and microscopic slide-coverslip (SL). Concentration results obtained with CASA were verified by manual counting on a Bürker hemocytometer (BH). No significant differences were found between the concentrations determined with BH vs. LJ and SL, whereas higher (p<0.01) values of this parameter were obtained with MK. Compared to MK and SL, significantly higher values were recorded in LJ for velocity (VCL and VAP) as well as amplitude of the lateral head displacement (ALH) and beat cross frequency (BCF), which was associated with significantly higher percentages of motile, progressively motile and rapidly progressive motile spermatozoa. Higher values for the linearity (LIN) and straightness (STR) of sperm movement were obtained for the analysis performed in MK and SL. In both these chambers, the results of all the linearity and kinetic parameters of sperm were similar (p>0.05). The results obtained show that CASA assessment of boar semen should account for the effect of counting chamber on the results of sperm motility and concentration, which confirms the need for further study on standardizing the automatic analysis of boar semen.

Validation of the sperm class analyser CASA system for sperm counting in a busy diagnostic semen analysis laboratory.

Sperm counts have been linked to several fertility outcomes making them an essential parameter of semen analysis. It has become increasingly recognised that Computer-Assisted Semen Analysis (CASA) provides improved precision over manual methods but that systems are seldom validated robustly for use. The objective of this study was to gather the evidence to validate or reject the Sperm Class Analyser (SCA) as a tool for routine sperm counting in a busy laboratory setting. The criteria examined were comparison with the Improved Neubauer and Leja 20-µm chambers, within and between field precision, sperm concentration linearity from a stock diluted in semen and media, accuracy against internal and external quality material, assessment of uneven flow effects and a receiver operating characteristic (ROC) analysis to predict fertility in comparison with the Neubauer method. This work demonstrates that SCA CASA technology is not a standalone 'black box', but rather a tool for well-trained staff that allows rapid, high-number sperm counting providing errors are identified and corrected. The system will produce accurate, linear, precise results, with less analytical variance than manual methods that correlate well against the Improved Neubauer chamber. The system provides superior predictive potential for diagnosing fertility problems.

Image cytometer method for automated assessment of human spermatozoa concentration.

In the basic clinical work-up of infertile couples, a semen analysis is mandatory and the sperm concentration is one of the most essential variables to be determined. Sperm concentration is usually assessed by manual counting using a haemocytometer and is hence labour intensive and may be subjected to investigator bias. Here we show that image cytometry can be used to accurately measure the sperm concentration of human semen samples with great ease and reproducibility. The impact of several factors (pipetting, mixing, round cell content, sperm concentration), which can influence the read-out as well as inter-operator and -cytometer variation on two different image cytometers (NC-3000 and SP-100) were evaluated. Furthermore, 725 semen samples were assessed both by manual assessment (WHO recommended method) and by image cytometry and tight correlations between the measured concentrations were shown. Moreover, by evaluation of repeated measurements it appeared that image cytometry produced more consistent and accurate measurements than manual counting of human spermatozoa concentration. In conclusion, image cytometry provides an appealing substitute of manual counting by providing reliable, robust and easy measurement of human sperm concentration.

[Influence of the depth of the sperm counting chamber on sperm motility].

OBJECTIVE: To investigate the influence of the depth of the sperm counting chamber on sperm motility. METHODS: We measured the depths of sperm counting chambers using the Filmetrics F20 Spectral Reflectance Thin-Film Measurement System. Then, according to the WHO5 manual, we analyzed 36 semen samples for the percentages of progressively motile sperm (PR) and non-progressively motile sperm (NP) and sperm motility (PR + NP) with the Ruiqi CFT-9201 computer-aided sperm analysis system, and compared the results of analysis. RESULTS: The depths of the 4 sperm counting chambers were 9.8, 12.7, 15.7 and 19.9 microm, respectively, and the obtained PR were (44.00 +/- 11.63), (41.96 +/- 12.62), (40.86 +/- 11.71) and (37.78 +/- 11.38)%, NP (13.54 +/- 3.01), (14.13 +/- 2.94), (14.91 +/- 3.02) and (16.53 +/- 2.77)%, and sperm motility (57.53 +/- 11.06), (56.08 +/- 11.97), (55.78 +/- 11.55) and (54.31 +/- 12.11)% from the 4 chambers, respectively. The depth of the sperm counting chamber was correlated negatively with PR (r = -0.993, P < 0.05) and sperm motility (r = -0.978, P < 0.05), but positively with NP (r = 0.989, P < 0.05). There were statistically significant differences between the 9.8 microm and 19.9 microm deep chambers in PR and NP (P < 0.05) though not in sperm motility among the 4 chambers of different depths. CONCLUSION: The impact of the depth of the sperm counting chamber on sperm motility should not be ignored, for the deviation of the results from the chambers of different depths may lead clinicians to incorrect diagnosis and consequently inappropriate therapeutic approaches. Different reference ranges of sperm motility need to be normalized in correspondence to the depths of sperm counting chambers.