ORIGINAL

 

Comparison of sperm concentration using the Makler and the Neubauer counting chambers

 

Cardona-Maya W, Berdugo J, Cadavid A

 

Reproduction Group. University of Antioquia. Medellín (Colombia)

 

Actas Urol Esp. 2008;32(4):443-445

 

 

Sperm concentration is one of the most important parameters in the evaluation of male fertility. Traditionally, sperms counts have been made with the Neubauer^® chamber, which has become the gold standard and is the method recommended by the World Health Organization (WHO)^1,2. However, other chambers have also been developed, such as the Makler^® (Sefi Medical Instruments, Haifa, Israel), Microcell^® (Concepción Technologies, Natick, MA, USA) and Horwell^® chamber (Horwell Ltd., London, UK), for determining sperm concentration. The advantage of the new chambers over the Neubauer^® chamber is that they use undiluted samples – thereby increasing the precision of the procedure and the reproducibility of the results. However, the introduction of these alternative chambers for the determination of sperm concentration has generated controversy, due to the reporting of non-uniform results when using the Makler^® chamber. It is important to compare the results obtained with different chambers, and to select the method best suited to each laboratory. The present study examines concordance between the sperm counts obtained with the Makler^® and Neubauer^® chambers in application to ejaculates of fertile males.

 

MATERIALS AND METHODS

Semen samples

Samples were collected from 112 males of confirmed fertility programmed for vasectomy in the Clínica Profamilia (Medellín, Colombia). Informed consent was obtained from all participants, and the study was approved by the ethics committee of the Medical Research Center of the University of Antioquia. The samples were collected by masturbation after 3‑5 days of sexual abstinence, in disposable sterile containers. Sperm concentration was determined twice, using each chamber, and if the difference between two counts of each chamber was over 10%, the counts were repeated.

 

Makler^® chamber

The grid of the Makler^® chamber measures 1 x 1 mm, with a depth of only 0.01 mm, and is subdivided into 100 squares, each measuring 0.1 x 0.1 mm. Thus, the total volume in the Makler^® chamber is 0.01 µl, or equivalent to 1/10 that of the Neubauer^® chamber. Sperm counting was made following the instructions of the manufacturer. A volume of 3-5 µl of semen sample previously immobilized by incubation at 60ºC for 10 minutes was transferred to the chamber, and counting was performed after 5 minutes in 10 squares of the chamber. The total sperm count is the end concentration expressed as 10⁶ spermatozoa/ml.

 

Neubauer^® chamber

The Neubauer^® chamber has a depth of 0.1 mm. It consists of 25 large squares and 16 small squares; each large square measures 1 x 1 mm in size. Preliminary visual calculation was made of the sperm concentration via microscopic observation of the undiluted sample, before preparing the chamber. The samples were diluted in HAM F-12 medium (Sigma Chemical Company, St. Louis, MO, USA), based on the visual calculation. The samples with a probable concentration of <15 x 10⁶ spermatozoa/ml were diluted 1:5; samples of 15-40 x 10⁶ spermatozoa/ml were diluted 1:10; samples of 40-200 x 10⁶ spermatozoa/ml were diluted 1:20; and samples of >200 x 10⁶ spermatozoa/ml were diluted 1:50. The chamber was filled with the sperm suspension, and counting was carried out using a light microscope. The end concentration was calculated according to the indications of the WHO².

 

Statistical analysis

The results relating to sperm concentration are expressed as the mean ± SD (standard deviation). The limits of concordance were deduced calculating 2 x DS of the differences between the Makler^® and Neubauer^® chambers, and the dispersion diagrams of the difference between the chambers (Y-axis) and the mean of the two chambers (X-axis) were plotted to visualize the data, using the Bland-Altman method. On the other hand, we separated the samples under and over 40 x 10⁶ spermatozoa/ml, and evaluated the statistical difference and correlation using the Student t-test and Pearson test, respectively. The results were analyzed using the Prism^® 4.0 program.

 

RESULTS

The mean sperm counts were 107.8 ± 73.64 and 106.2 ± 74.24 x 10⁶ spermatozoa/ml using the Makler^® and Neubauer^® chambers, respectively. The Bland-Altman plot indicated that the intervals obtained with either chamber were similar, with a bias of -1.55 and a 95% confidence interval of between -46.8 and 43.7 (Fig. 1).

 

FIGURE 1. Correlation between the sperm counts obtained in 112 fertile males using the Makler^® and Neubauer^® chambers.
The difference between the sperm counts using the Makler^® and Neubauer^® chambers (Y-axis) is plotted against the mean of the two counts (X-axis). The diagram shows the magnitude of the difference between the two chambers and the distribution close to zero.

 

In addition, the comparison of the samples under and over 40 x 10⁶ spermatozoa/ml using the Makler^® and Neubauer^® chambers was not statistically significant (p>0.05), and the Pearson correlation coefficient was high (sample under 40 x 10⁶ = 0.88, and samples over 40 x 10⁶ = 0.93).

 

DISCUSSION

The Makler^® chamber for determining sperm concentration requires a single aliquot of the original sample, and is easier to use than the Neubauer^® chamber – affording a calculation that comes close to the true sperm concentration present. Variability in the determination of sperm concentration between the different chambers is inevitable, and the differences have been attributed to dilution of the semen samples, the chambers used to count the sperm, sperm sample properties such as viscosity, and differences among the technicians performing the procedure³. Thus, research in this field is justified with the purpose of validating one of the existing methods, or for designing new chambers capable of offering less variable results⁴.

No significant differences were found between the Makler^® and Neubauer^® chambers in performing the sperm counts. This does not coincide with the observations of other authors, however. In effect, Ginsburg and Armant⁵, in a study of 10 individuals, observed a statistically significant 62% increase on comparing the counts obtained with the Makler^® chamber versus the Neubauer^® chamber. Christensen et al.⁶ obtained similar results in application to bull sperm. In turn, Imade et al.⁷ reported significant differences in a study of 50 samples. On the other hand, Matson et al.⁸ showed that these differences could be reduced by carefully following the instructions of the manufacturer.

In contrast, our results coincide with those published by Sukcharoen et al.⁹ and Mahmoud et al.³, who found no statistically significant differences (p>0.05) between the two chambers. In addition, Sukcharoen et al.⁹ found that in samples with concentrations under 40 x 10⁶ spermatozoa/ml, the count using the Makler^® chamber was greater (p<0.0001) than in samples with a larger concentration of sperm. This may suggest that the Makler^® chamber is inadequate for samples of this kind. Therefore, we divided the samples under and over this critical value, and found the count obtained with each chamber to be similar in both sample groups (p>0.05). This difference could be explained by the number of samples used by Sukcharoen et al.⁹ (n=55), compared with the 112 ejaculates in our series – 92 of which corresponded to the group with concentrations of under 40 x 10⁶ spermatozoa/ml.

There is no agreement on the protocol to be used in the test recommended by the WHO as the gold standard for sperm counting². Each laboratory must experiment with its own population in order to determine which chamber is most adequate. However, it must be pointed out that the Makler^® chamber is rapid and simple to use. In sum, our results show that both chambers are precise, even when used in application to samples with low counts.

 

Acknowledgements

The authors thank the Clínica Profamilia (Medellín, Colombia).

This study was supported by the University of Antioquia, and Cardona-Maya W. receives financial support from COLCIENCIAS.

 

REFERENCES

1. OMS. Manual de Laboratorio de la OMS para el examen del semen humano y de la interacción entre el semen y el moco cervical. Bogotá,: Editorial panamericana; 1992.

2. WHO. WHO Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction. Cambridge: Cambridge University Press; 1999.

3. Mahmoud AM, Depoorter B, Piens N, Comhaire FH. The performance of 10 different methods for the estimation of sperm concentration. Fertil Steril. 1997;68(2):340-345. [PubMed]

4. Coetzee K, Menkveld R. Validation of a new disposable counting chamber. Arch Androl. 2001;47(2):153-156. [PubMed]

5. Ginsburg KA, Armant DR. The influence of chamber characteristics on the reliability of sperm concentration and movement measurements obtained by manual and videomicrographic analysis. Fertil Steril. 1990;53(5):882-887. [PubMed]

6. Christensen P, Stryhn H, Hansen C. Discrepancies in the determination of sperm concentration using Burker-Turk, Thoma and Makler counting chambers. Theriogenology. 2005;63(4):992-1003. [PubMed]

7. Imade GE, Towobola OA, Sagay AS, Otubu JA. Discrepancies in sperm count using improved Neubauer, Makler, and Horwells counting chambers. Arch Androl. 1993;31(1):17-22. [PubMed]

8. Matson P, Irving J, Zuvela E, Hughes R. Delay in the application of the cover glass is a potential source of error with the Makler Counting Chamber. Fertil Steril. 1999;72(3):559-561. [PubMed]

9. Sukcharoen N, Ngeamjirawat J, Chanprasit Y, Aribarg A. A comparison of Makler counting chamber and improved Neubauer hemocytometer in sperm concentration measurement. J Med Assoc Thai. 1994;77(9):471-6. [PubMed]

 

Correspondence author: Dr.  Walter Cardona-Maya

Grupo Reproducción. Universidad de Antioquia -SIU-, Medellín.

Carrera 53 # 61-30, Colombia. Tel.: 57 (4) 2196685

Author e-mail: wdcmaya@yahoo.com/reproduccion@medicina.udea.edu.co

http://reproduccion.udea.edu.co/

Papel information: Original – Andrology - infertility

Manuscript received: june 2007

Manuscript accepted: july 2007