Abstract
According to the World Health Organization (WHO) manual, sperm concentration should be measured using an improved Neubauer hemocytometer, while sperm motility should be measured by manual assessment. However, in China, thousands of laboratories do not use the improved Neubauer hemocytometer or method; instead, the Makler counting chamber is one of the most widely used chambers. To study sources of error that could impact the measurement of the apparent concentration and motility of sperm using the Makler counting chamber and to verify its accuracy for clinical application, 67 semen samples from patients attending the Department of Andrology, West China Second University Hospital, Sichuan University (Chengdu, China) between 13 September 2023 and 27 September 2023, were included. Compared with applying the cover glass immediately, delaying the application of the cover glass for 5 s, 10 s, and 30 s resulted in average increases in the sperm concentration of 30.3%, 74.1%, and 107.5%, respectively (all P < 0.0001) and in the progressive motility (PR) of 17.7%, 30.8%, and 39.6%, respectively (all P < 0.0001). However, when the semen specimens were fixed with formaldehyde, a delay in the application of the cover glass for 5 s, 10 s, and 30 s resulted in an average increase in the sperm concentration of 6.7%, 10.8%, and 14.6%, respectively, compared with immediate application of the cover glass. The accumulation of motile sperm due to delays in the application of the cover glass is a significant source of error with the Makler counting chamber and should be avoided.
Keywords: concentration, improved Neubauer hemocytometer, Makler counting chamber, motility, sperm
INTRODUCTION
Semen analysis plays a crucial role in assessing male fertility for couples facing difficulties in conceiving.1 The sperm concentration is closely associated with the time it takes for pregnancy to occur and the success rate of pregnancy, making it a valuable predictor of conception. To determine the sperm concentration accurately, the World Health Organization (WHO) manual recommends using an improved Neubauer hemocytometer, which is considered the most reliable method available.2 However, the hemocytometer is not suitable for routine measurement of sperm concentration due to its labor-intensive and time-consuming nature.3 To address this issue, the Makler counting chamber was invented in 1978, and since then, an increasing number of laboratories have embraced this technology. The Makler counting chamber has a depth of 10 μm, making it ideal for detecting both sperm concentration and motility efficiently and conveniently.4,5 This chamber offers a faster and more practical alternative to the traditional hemocytometer, allowing for more efficient analysis of sperm samples in routine laboratory settings.
The use of Makler counting chambers has several advantages. First, given the size of the sperm and the depth of the Makler chamber, the formation of a monolayer is the main characteristic of this chamber, which makes the Makler chamber the best instrument for detecting sperm concentration and motility. Second, it allows direct testing without the need to dilute samples. This approach is beneficial because it simplifies the testing process and saves time. Third, the Makler chamber enables the simultaneous detection of sperm concentration and motility. This feature is crucial because it provides a comprehensive analysis of sperm quality in a single test. Fourth, it allows for rapid and objective detection through computer-aided sperm analysis (CASA). This technology ensures accurate and reliable results. Despite these advantages, several studies have shown that the sperm concentration and motility results obtained using the Makler counting chamber are relatively high compared to those of other sperm counting chambers.6,7,8,9,10,11,12,13,14,15,16 This discrepancy raises concerns about the accuracy and reliability of the Makler counting chamber. In fact, the WHO manual has never included the Makler counting chamber in the list of recommended sperm counting chambers. Notably, there is a lack of research on the factors that may influence sperm analysis when using the Makler counting chamber. Whether analyzing latex beads or samples of human or animal semen, very few studies have focused on investigating the potential factors that could affect the accuracy of the results obtained with this specific counting chamber.17,18,19 Therefore, further research is needed to better understand the performance and limitations of the Makler counting chamber to ensure reliable and accurate sperm analysis.
We demonstrated that a delay in the application of the cover glass is a key determinant that can greatly influence the accuracy of the Makler counting chamber, primarily in relation to the accumulation of motile sperm. Our study highlights crucial factors that are often disregarded and underestimated by numerous technicians in their daily practices. Moreover, this study offers substantial evidence that attests to the exceptional accuracy and clinical relevance of the Makler counting chamber. Meticulous scientific experiments and rigorous data analysis confirmed the reliability and validity of this device in various clinical applications. This evidence further strengthens the confidence of health-care professionals and encourages widespread adoption of the Makler counting chamber in diagnostic and treatment procedures.
MATERIALS AND METHODS
Samples
Approval was obtained from the Institutional Review Board of West China Second University Hospital, Sichuan University (Chengdu, China; Approval No. 2023072). A total of 67 semen samples with a volume ≥2 ml, sperm concentration ≥16 × 106 ml−1, progressive motility (PR) ≥5%, and normal viscosity were obtained by masturbation after a minimum of 2 days and a maximum of 7 days of ejaculatory abstinence from patients attending the Department of Andrology, West China Second University Hospital, Sichuan University between 13 September 2023 and 27 September 2023. Informed consent was obtained from all patients.
Delays in the application of the cover glass with the Makler counting chamber
First, we investigated the impact of delays in the application of cover glass with a Makler counting chamber (Sefi Medical Instruments, Haifa, Israel) on sperm concentration and PR in 21 fresh semen samples. A 5 μl drop of liquefied semen was placed in the center of the lower platform of the chamber, and the cover glass was applied either immediately (almost 0 s) or after a delay of 5 s, 10 s, or 30 s. The motility/concentration module of the CASA system (Suijia Software Co., Ltd., Beijing, China) was used to assess and compare the sperm concentration and PR.
Furthermore, we sought to compare the effects of delaying the application of the cover glass of the Makler counting chamber on the sperm concentration in 14 semen samples after formaldehyde fixation. A 5 μl drop of 0.1% formaldehyde-fixed semen was placed in the center of the lower platform of the chamber, and the cover glass was applied either immediately (almost 0 s) or after a delay of 5 s, 10 s, and 30 s. The motility/concentration module of the CASA system was used to assess and compare the sperm concentration.
Comparison of the Makler counting chamber with immediate application of the cover glass with other chambers
Considering that the improved Neubauer hemocytometer is recommended by the WHO manual for the detection of sperm concentration, we aimed to evaluate the detection efficacy of the Makler counting chamber in relation to the improved Neubauer hemocytometer (Paul Marienfeld Gmbh, Lauda-Konigshofen, Germany) on sperm concentration in 22 fresh semen samples. By comparing the sperm concentration detection results obtained from both chambers, we were able to assess differences in accuracy between the two methods. An improved Neubauer hemocytometer was used to detect the sperm concentration in strict accordance with the 6th edition of the WHO manual.2
We expanded our study to include a comparison between a Makler counting chamber and a disposable commercially available capillary-filled slide (Suijia Software Co., Ltd.) in terms of sperm concentration and PR in 23 fresh semen samples. A 5 μl drop of liquefied semen was placed in the loading area of the capillary-filled slide, and after waiting 30 s for the semen to be evenly distributed in the chamber at a depth of 10 μm, the sperm concentration and motility were measured by CASA. Furthermore, we compared the sperm concentration in 30 fresh semen samples after applying the cover glass immediately with that after formaldehyde fixation.
In addition, we manually evaluated and compared the PR results of the Makler counting chamber with those of fresh wet preparation slides, as recommended by the WHO manual in 22 fresh semen samples, to assess the accuracy of the PR measured by the Makler counting chamber.2
Quality assurance
The research was performed at the Department of Andrology, West China Second University Hospital, Sichuan University, which obtained an International Organization for Standardization (ISO) 9001:2015 Certification. The laboratory has established a complete internal quality control scheme for sperm concentration and motility detection. Technicians underwent standardized training in semen analysis; the variability within and between technicians was assessed and accepted, and the test results were repeated to avoid sampling errors.
Statistical analyses
Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). The normality of the differences was tested using the Shapiro‒Wilk test. Comparisons between groups were analyzed using one-way analysis of variance (ANOVA) and Tukey’s multiple comparison. P < 0.05 was considered statistical significance. P-trend was used in regression analysis to identify the linear trend relationship between the independent variable and the dependent variable. The paired t-test and Bland‒Altman plots were used to assess the pairs of results for agreement.
RESULTS
Delays in the application of the cover glass with the Makler counting chamber
For fresh samples with motile sperm, a delayed application of the cover glass led to a significant increase in sperm concentration and forward motility. First, there was a progressive increase in sperm concentration and PR as the delay in applying the cover glass increased (Figure 1a and 1b). Compared with applying the cover glass immediately, a delay in applying the cover glass of 5 s, 10 s, and 30 s resulted in an average increase in sperm concentration of 30.3%, 74.1%, and 107.5%, respectively, and an increase in sperm PR of 17.7%, 30.8%, and 39.6%, respectively (Table 1).
Figure 1.
The influence of different delays in the application of the cover glass on the test results. (a) Sperm concentrations of fresh samples were detected after a delay in cover application of 0 s, 5 s, 10 s, and 30 s, respectively (n = 21). (b) Sperm PR of fresh samples were detected after a delay in cover application of 0 s, 5 s, 10 s, and 30 s, respectively (n = 21). (c) Sperm concentrations of fixed samples were detected after a delay in cover application of 0 s, 5 s, 10 s, and 30 s, respectively (n = 14). The results are expressed as the mean ± standard deviation (one-way ANOVA). *P < 0.05, **P < 0.01, ****P < 0.0001. ANOVA: analysis of variance; PR: progressive motility; NS: not significant.
Table 1.
Sperm concentration and progressive motility in fresh samples measured using a Makler counting chamber (n=21)
| Variable | Delay in applying cover (s) | P-trendd | |||
|---|---|---|---|---|---|
|
| |||||
| 0 | 5 | 10 | 30 | ||
| Sperm concentration (×106 ml−1), mean±s.d. | 68.2±33.6 | 88.9±40.8a | 118.7±54.9a,b | 141.5±65.8a,b,c | <0.0001 |
| Sperm concentration increase rate (compared to 0 s), % | 0 | 30.3 | 74.1 | 107.5 | |
| Sperm PR (%), mean±s.d. | 48.5±17.9 | 57.0±19.8a | 63.4±19.5a,b | 67.7±19.4a,b,c | 0.0035 |
| Sperm PR increase rate (compared to 0 s), % | 0 | 17.7 | 30.8 | 39.6 | |
aP<0.001, the marked groups versus 0 s; bP<0.001, the marked groups versus 5 s; cP<0.001, the marked groups versus 10 s; dP-trend, P value of the linear trend test. PR: progressive motility; s.d.: standard deviation
When the semen specimens were fixed with formaldehyde, the delayed application of the cover slip resulted in only a small increase in sperm concentration (Figure 1c). Compared with applying the cover glass immediately, a delay in applying the cover glass of 5 s, 10 s, and 30 s resulted in an average increase in sperm concentration of 6.7%, 10.8%, and 14.6%, respectively (Table 2).
Table 2.
Sperm concentration in fixed semen samples measured using a Makler counting chamber (n=14)
| Variable | Delay in applying cover (s) | P-trendd | |||
|---|---|---|---|---|---|
|
| |||||
| 0 | 5 | 10 | 30 | ||
| Sperm concentration (×106 ml−1), mean±s.d. | 67.6±36.4 | 72.1±41.0 | 74.9±42.2a | 77.4±43.5a,b,c | 0.55 |
| Sperm concentration increase rate (compared to 0 s), % | 0 | 6.7 | 10.8 | 14.6 | |
aP<0.001, the marked groups versus 0 s; bP<0.001, the marked groups versus 5 s; cP<0.001, the marked groups versus 10 s; dP-trend, P value of the linear trend test. s.d.: standard deviation
In addition, we divided the samples into two groups according to the threshold of 50% PR. In the PR ≥50% group, a delay in applying the cover glass of 5 s, 10 s, and 30 s resulted in an average increase in sperm concentration of 35.8%, 85.4%, and 125.6%, respectively; while in the PR <50% group, a delay in applying the cover glass of 5 s, 10 s, and 30 s resulted in an average increase in sperm concentration of 22.4%, 57.9%, and 81.5%, respectively (Supplementary Table 1). The greater the sperm PR, the higher the proportion of increased sperm concentration caused by a delay in the application of the cover (Figure 2).
Supplementary Table 1.
Concentration of sperm in fresh samples with progressive motility ≥50% (n=12) and progressive motility <50% (n=9) measured using the Makler counting chamber
| Delay in applying cover (s) | Sperm concentration (×106/ml) | Sperm concentration increase rate (compared to 0 s) | ||
|---|---|---|---|---|
|
|
|
|||
| PR ≥50% | PR <50% | PR ≥50% | PR <50% | |
| 0 | 70.3±25.6 | 65.4±43.7 | - | - |
| 5 | 95.4±29.3a | 80.1±53.1d | 35.8% | 22.4% |
| 10 | 130.3±41.3a,b | 103.3±68.7d,e | 85.4% | 57.9% |
| 30 | 158.5±52.6a,b,c | 118.8±77.4d,e,f | 125.6% | 81.5% |
| P-trendg | <0.0001 | 0.071 | - | - |
aP<0.001 (vs 0 s); bP<0.001 (vs 5 s); cP<0.001 (vs 10 s); dP<0.05 (vs 0 s); eP<0.05 (vs 5 s); fP<0.05 (vs 10 s); gP-trend is P value of the linear trend test. Values are means±s.d. PR: progressive motility; s.d.: standard deviation
Figure 2.
The influence of different delays (0 s, 5 s, 10 s, and 30 s) in the application of the cover glass on the sperm concentration detection in samples with different PR. The results are expressed as the mean ± standard deviation (one-way ANOVA). *P < 0.05, ****P < 0.0001. PR: progressive motility; ANOVA: analysis of variance.
Comparison of Makler counting chamber with immediate application of the cover glass with other chambers
The Makler counting chamber showed no significant difference in sperm concentration between immediately covered fresh samples and those fixed with formaldehyde, indicating good consistency (mean ± standard deviation [s.d.]: 68.8 × 106 ± 40.3 × 106 ml−1 vs 64.5 × 106 ± 39.0 × 106 ml−1, P = 0.06; Figure 3a).
Figure 3.
Bland–Altman diagram showing the plot of the difference between the results of (a) sperm concentration (×106 ml−1) in fresh and fixed semen against the average of the pair (n = 30), (b) sperm concentration (×106 ml−1) in the Makler chamber and improved Neubauer counting chamber against the average of the pair (n = 22), (c) sperm concentration (×106 ml−1) in the Makler chamber and disposable counting chamber against the average of the pair (n = 23), (d) sperm PR (%) in the Makler chamber and disposable counting chamber against the average of the pair (n = 23), and (e) sperm PR (%) in the Makler chamber and fresh wet preparation slide against the average of the pair (n = 22). The red dotted lines show the limits of agreement. The green dotted line shows the mean value of the differences. The black dotted line is the zero line used to assess the discrepancy of the observed mean difference from zero. The diagram shows that more than 95% of the differences in the studied subjects lay within the limits of agreement, indicating acceptable reliability between the test pairs. s.d.: standard deviation; PR: progressive motility.
When the cover was immediately applied, the sperm concentration measured by the Makler counting chamber showed no significant difference compared to the improved Neubauer counting chamber (mean ± s.d.: 56.0 × 106 ± 15.1 × 106 ml−1 vs 56.1 × 106 ± 15.9 × 106 ml−1, P = 0.96; Figure 3b). Furthermore, there was no significant difference in sperm concentration (mean ± s.d.: 76.4 × 106 ± 53.6 × 106 ml−1 vs 75.9 × 106 ± 51.3 × 106 ml−1, P = 0.71; Figure 3c) or PR (mean ± s.d.: 55.8% ± 14.1% vs 54.0% ± 13.2%, P = 0.06; Figure 3d) measured by the Makler counting chamber compared to the disposable capillary-filled slide.
In addition, there was no significant difference in sperm PR measured by the Makler counting chamber compared to the fresh wet preparation slide (mean ± s.d.: 50.0% ± 19.7% vs 49.6% ± 17.7%, P = 0.78; Figure 3e).
DISCUSSION
The results of this study revealed that in the presence of motile sperm, delayed application of the cover glass had a significant impact on the accuracy and reliability of the detection results of the Makler counting chamber, which led to false increases in sperm concentration and PR. As the delay in applying the cover glass increased, there was a noticeable trend of progressive increase in both sperm concentration and motility. A previous research showed that there was a progressive increase in concentration in both sperm and beads with a longer delay in applying the cover glass, which was attributed to the settling of cells or particles before the suspending medium was distributed.18 However, Dr. Makler calculated the subsidence rate and repeated the steps exactly and found that the increments in both the calculations and experiments were considerably lower than those in Matson’s results.19 Unlike previous studies, through multiple semen samples, we found that a delay in the application of the cover glass led to a significant increase in sperm concentration only in samples with motile sperm. In samples with immotile sperm, this phenomenon is not obvious. A previous research on the Makler counting chamber has suggested that a delay in the application of the cover glass led to a significant increase in sperm concentration due to sperm settling. However, in this study, through the analysis of fresh semen samples and fixed immotile sperm samples with delayed covering, settling factors led to a slight increase in sperm concentration and can, therefore, be excluded as a factor causing the significant increase in sperm concentration due to delayed covering. Similarly, based on previous research and our data, volatility can also be ruled out as a factor.17,18 Interestingly, we observed a positive correlation between the proportion of PR and the magnitude of sperm concentration increase caused by delayed covering. This result suggests that there are certain factors that rapidly and significantly lead to the accumulation of motile sperm during the short delay in covering. With the development of computer technology, computational fluid dynamics (CFD) methods have been widely applied in the study of sperm navigation mechanisms, including chemotaxis, thermotaxis, rheotaxis, and near-wall effects.20,21,22,23 Chemotaxis refers to the ability of sperm to sense and bind to certain chemotactic molecules, causing them to move toward or away from the chemotactic source.20 Thermotaxis is the ability of sperm to sense the temperature of their surroundings and swim toward higher temperatures.21 Rheotaxis is a characteristic of sperm that senses the flow state of the surrounding fluid and uses it to regulate its movement; sperm exhibit a negative rheotaxis that flows upstream.22 Near-wall effects describe the tendency of sperm to swim close to the wall when swimming in the bounded area, so that sperm can rely on the wall to guide the direction of swimming in the reproductive tract.23 However, it appears that none of the previously mentioned studies on mechanisms have provided a comprehensive and convincing rationale to account for this phenomenon. Here, we propose a hypothesis that spermatozoa are evenly distributed along with droplets after immediate covering. However, motile sperm tend to swim to the middle region when the cover is not placed immediately. The longer the delay in covering is, the more motile sperm will accumulate in the middle region. When the cover glass is applied, the liquid is forced to diffuse to the periphery, and the sperm has no time to change the direction of movement through its own movement (the effect of the velocity vector is still within the range of the original droplet), resulting in increased sperm concentration and PR in the central counting area (Figure 4).
Figure 4.
Cross-section and microscopic diagram of the Makler counting chamber. Immediately after the application of the cover, the sperm distribution was uniform. After a delay in application, the accumulation of motile sperm results in uneven sperm distribution.
Most previous studies showed that the Makler counting chamber results in higher sperm concentrations and/or PR than other types of chambers, including the improved Neubauer hemocytometer, and there were a limited number of studies showing good consistency between the Makler counting chamber and the hemocytometer.24,25,26 However, few studies have explored and elaborated the causes and solutions for the overestimated results of the Makler counting chamber. The improved Neubauer hemocytometer is recommended by the WHO to detect sperm concentration. However, our comparative data showed that after conducting several tests, we found no significant difference in sperm concentration between the Makler counting chamber and the improved Neubauer hemocytometer. This result suggested that both methods are equally effective in accurately determining the sperm concentration in a sample. In addition, there was no significant difference in PR between the Makler counting chamber and the fresh wet preparation slide suggested by the WHO manual. Furthermore, when comparing the Makler counting chamber with disposable commercially available capillary-filled slides for samples of normal viscosity, we also observed no significant difference in either sperm concentration or PR. These findings suggested that the Makler counting chamber can yield similar results to the commonly used slides or recommended counting chamber, provided standardized operation procedures are followed, including the immediate application of the cover to prevent any potential alterations in the sample. Based on the information provided in the present study, it can be inferred that certain studies might have overlooked the influence of the delayed application of the cover glass on the enhanced detection outcomes when utilizing Makler counting chambers. When testing immotile sperm samples, a delay in the application of the cover had little effect on concentration. This suggested that another important issue that should not be overlooked is the sperm concentration external quality assessment (EQA) scheme, regardless of whether beads or fixed sperm are used; when the Makler counting chamber was used, there was no noticeable impact on the test results. Therefore, performing EQA cannot effectively detect any potential problems arising from a delay in covering in the laboratory.
Based on the findings of our comprehensive questionnaire survey and the results obtained from the EQA approach, an overwhelming majority of clinical laboratories in China have moved away from utilizing the improved Neubauer hemocytometer and fresh wet preparation slide for the purpose of sperm concentration and motility detection, although these methods are recommended by the WHO manual. Instead, the most commonly used method is disposable capillary-loaded slides, followed by the Makler counting chamber. This outcome is not surprising considering the widespread implementation of CASA systems in China. The improved Neubauer hemocytometer is a time-consuming and cumbersome process; it lacks the capability to simultaneously assess sperm motility and cannot be used with CASA. Strict adherence to standard operation procedure, including immediate application of the cover, demonstrated that the Makler counting chamber has accurate concentration detection results comparable with those of the improved Neubauer hemocytometer. Given its ability to simultaneously measure sperm concentration, motility, and compatibility with CASA, the Makler counting chamber is feasible for clinical laboratory testing. Given the current situation in China, it is imperative to identify and correct potential sources of error in the use of the Makler counting chamber.
In summary, the delay in applying the cover leads to the accumulation of motile sperm when using the Makler counting chamber. This is one of the main factors leading to significant errors in overestimating sperm concentration and motility. Therefore, it is crucial to strictly control the time of cover application and ensure immediate detection after loading the sample. In the Laboratory of Andrology, West China Second University Hospital, Sichuan University, we have established a rigorous training program for our technicians to ensure they have the necessary skills and expertise to use the Makler counting chamber effectively. Additionally, we prioritize the regular implementation of quality assurance assessments to ensure precise results. Unfortunately, we have come across other laboratories that do not adhere to these rigorous practices, leading to significant issues in the proper use of Makler counting chambers. These problems can result in substantial errors in the detection of spermatozoa, emphasizing the crucial importance of well-trained technicians in this process. When problems arise during the utilization of the Makler counting chamber, it is crucial for the technician to perform separate counting and motility assessments. Separating these two evaluations allows for a more accurate and reliable understanding of the sperm sample’s characteristics. Specifically, when counting spermatozoa, immobilization is essential to obtain precise results.
AUTHOR CONTRIBUTIONS
LY conducted the experiment and statistical analysis and wrote the draft. QYC provided drawing assistance and participated in the experimental operation. YLJ, YZ, and TTY participated in the experimental operation. YBW participated in the experimental operation, data collection, and validation. FPL put forward experimental ideas and reviewed and revised the paper. All the authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
ACKNOWLEDGMENTS
We thank Engineer Hai-Feng Wang (China Mobile Communications Group Co., Ltd.) for providing knowledge of fluid mechanics and statistical physics, who has no competing interests in this study. This study was supported by the Natural Science Foundation of China (No. 32171264 and No. 81974226) and the Sichuan Science and Technology Program (2023NSFSC1609).
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
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