JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):51
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263590

P-39. Comparative Analysis of Sperm Concentration in Human Samples Using Makler and Neubauer Chambers

Giovana Vitirito¹, Camila Nogueira², Eduardo Mancini Gomes Costa¹, Gabrielly Alves Rodrigues Silva¹, Monique Helen Nogueira Pádua¹, Valéria Barradas¹, Paula Intasqui¹

¹Human Reproduction Section, Division of Urology, Universidade Federal de São Paulo - São Paulo - SP - Brasil
² Associação Paulista Para o Desenvolvimento da Medicina - São Paulo - SP - Brasil

Objective: This study aimed to compare sperm concentration values obtained using Makler and Neubauer chambers and to assess the reproducibility of readings among five observers with varying degrees of experience.
Methods: A total of 21 semen samples were collected from volunteers over 18 years of age. Each sample was divided into two aliquots: one diluted in distilled water to immobilize spermatozoa for counting in the Neubauer chamber, and the other used directly in the Makler chamber with un-immobilized spermatozoa, simulating laboratory routine conditions. Neubauer chamber readings were standardized according to WHO recommendations, while the preparation of the Makler chamber followed guidance from two experienced professionals and information from selected articles in the literature, considering that there is no formal standardization for this method. To ensure consistency, all dilutions and chamber preparations were performed by the same person. Readings were conducted in a randomized and anonymized manner by previously trained observers. In order to compare different degrees of experience, 5 observers were recruited, each with different past uses of each chamber. Observers with many years of involvement in seminal analysis were considered experts, one with the Makler and Neubauer chambers and the other with the Neubauer chamber. The remaining 3 observers had been trained to use each chamber, but with less hands-on experience. Comparisons between counting chambers and between observers were performed using a generalized linear mixed model (GLMM) with logarithmic transformation, considering “chamber” and “observer” as fixed effects and “individual” as a random effect. To evaluate the error relative to the reference value, sperm concentration values obtained with the Neubauer chamber observed by two external experienced analysts were considered. A non-parametric test for repeated measures (Friedman) was applied.
Results: No statistically significant differences were observed between chambers (p=0.138) or between observers (p=0.189), indicating good reproducibility among the evaluators. However, analysis of error relative to the reference value revealed a significant difference between methods (χ²=4.20; p=0.040), with the Makler chamber showing a negative mean error (–4.687), suggesting underestimation of sperm concentration values, while the Neubauer chamber presented a mean error near to zero (0.267), closer to the reference value.
Conclusion: Although no statistically significant differences were observed between chambers or readers, the Makler chamber tends to underestimate sperm concentration, possibly due to the path the spermatozoa take in and out of the counting area. The Neubauer chamber, when used following WHO recommendations, benefits from a well-established protocol, which contributes to a greater uniformity and reliability of results. As a downside, it requires prior dilution with some calculation afterwards, longer handling time, and sperm immobilization. In contrast, the Makler chamber has the advantage of allowing direct counting without the need for sample dilution, which can streamline procedures in busy laboratory routines. However, the absence of standardized operating protocols and its susceptibility to errors, especially with motile spermatozoa, may compromise accuracy, therefore interfering with patient clinical management. These factors should be considered when selecting the most appropriate chamber for each specific laboratory context and in future proposals in standardization for the Makler chamber.