Commentary on “Delayed covering causes the accumulation of motile sperm, leading to overestimation of sperm concentration and motility with a Makler counting chamber”

The analysis of the ejaculate, better known as spermiogram, represents the first and main step to identify whether a series of sperm quality parameters are within the norm and therefore are consistent with normal sperm fertilizing capacity. Among these, sperm concentration and motility are the first parameters to be evaluated through an estimation carried out by expert examiners. Over the last four decades, the computer-aided sperm analysis (CASA) system has been progressively adopted in many andrological and assisted reproduction centers on the basis that the computer readings are more precise than the human eye so ensuring standardized and reproducible data. The computerized analysis is accurate. However, expert examiners have noted an overestimation of the observed values. This overestimation occurs not only because of excess sample volume but also due to fluctuations in the parameters, which seem to change continuously after multiple sequential microscope examinations (personal communication). Although the 5th edition of World Health Organization (WHO) manual recommends using the hemocytometer for sperm analysis, the Makler counting chamber is considered as the most reliable effective and accurate tool, in most of the andrological laboratories that perform spermiograms either with or without CASA system. Based on a 10-μm deep monolayer, the Makler chamber offers a clear and simultaneous view of sperm concentration and motility as cells are allowed to free horizontal movements but cannot overlap each other.1 Furthermore, the count can be obtained from a fresh sample and the grid with 10 × 10 squares offers an easy and fast way of counting. Compared with other counting chambers, the advantage of Makler has appeared since 1980s due to the pressed coverslip, especially for sperm motility evaluation.2 I read with interest the article by Yu et al.;3 they report an unexpected possible source of errors in using Makler counting chamber. The authors confirmed previous data,4 evidencing an overestimation of sperm concentration when the application of the cover glass on the small drop of liquefied ejaculate is delayed, respectively of 5 s, 10 s, and 30 s. On 67 seminal fluid samples monitored, the sperm concentration increase was rated 30%, 70%, and 107%, respectively, and forward motility rated 19%, 31%, and 42%, respectively, more than the initial reading carried out after the immediate application of the cover glass. The most interesting data were obtained by performing a comparison with the same sample fixed with formaldehyde because the immobility of spermatozoa resulted in a decreased percentage of both parameters’ overestimation. These data lead the authors to conclude that only motile spermatozoa tend to accumulate in the center of the chamber even in the shortest interval between the deposition of 10 μl of ejaculate and the placement of the cover. They explain this behavior as phenomenon of fluid physics where the lid application forces the plasma to diffuse to the periphery of the drop. Hence, it appears to be recommended to be very fast in applying the cover as well as fast to observe and count the sample. The authors conclude the study by proposing two important options: the use of fixative that immobilize spermatozoa to obtain precise and reliable concentration evaluation, and the coordination by expert biology operating simultaneously on the same semen sample. This is a short technical study, whose strength resides in an accurate training of specialized technicians and examiners. In fact, the “time factor” plays a fundamental role. However, this is also a weakness. In the daily routine of andrological centers, semen samples are analyzed in sequence, which prevents specific attention to each individual sample. Another factor of weakness comes from the question: does accumulation also continue after placement of the cover glass? This information may help in understanding the overestimation of sperm parameters observed by the CASA system. An expert examiner should always make an initial observation under the microscope before activating the software. This could cause those crucial seconds to pass and modify the real parameter evaluation. In my opinion, other two possible sources of error come from the incomplete liquefaction state of the ejaculate and from a bad sperm morphology. Anomalies related to motility, such as those affecting sperm tails, can cause the movements and trajectories of the sperm population to be non-uniform, adding an additional layer of confusion in the examination of the samples. I agree with the authors’ recommendation that the sample should be fixed for the most reliable evaluation. However, I am concerned about the use of potentially contaminating fixatives in a seminology laboratory, where capacitated seminal fluids are often prepared for in vivo inseminations, in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI). Furthermore, I believe that this time-consuming procedure may go into conflict with the workload and time factor of a laboratory that must examine several samples of seminal fluid per day. This study is an interesting indication and starting point to ensure a rigorous evaluation of the quality of seminal fluid. If possible and within time constraints, carrying out duplicates or triplicates of the test can help in data evaluation in research. This approach provides a statistically reliable average of the data, especially in clinical routines. COMPETING INTERESTS The author declares no competing interests.