Validation of sperm counting methods using limits of agreement.

A variety of methods exist for counting sperm. Since the introduction of semen analysis, one of these methods, the hemocytometer, has been regarded as the gold standard by andrology laboratories and the World Health Organization (WHO, 1999). The flexible features of this approach, involving fixation and immobilization of sperm, dilution of highly concentrated samples, and the counting of sperm in a single plane, contribute to the accuracy of the Improved Neubauer hemocytometer and its relative ease of use. This method is strongly accepted within andrology clinics and has been clinically validated by a number of studies (Dunphy et al, 1989; Tomlinson et al, 1996, 1999; Guzick et al, 2001). As technology and techniques improve, manufacturers are continually trying to develop newer, simpler, quicker and more accurate methods for determining sperm concentration. Busy assisted reproduction technology (ART) laboratories in particular would find a quicker yet comparatively accurate method highly desirable, since sperm counting is an essential part of the semen preparation process. Although modern methods may be faster, since unfixed, undiluted semen is used, some labs find these analyses more difficult to use and believe that the counting of motile sperm may produce erroneous results. Furthermore, the WHO states that the newly introduced methods ‘‘are convenient in that they can be used without dilution of the specimen, but that they may lack the accuracy of the hemocytometer technique especially for highly viscous and/or heterogeneous specimens. If such chambers are to be used, their adequate accuracy and precision must be established by comparison with hemocytometers’’ (WHO, 1999). In addition, it is now a requirement of laboratory accreditation systems that laboratories provide clinical validation for all methods used (ie, demonstrate that they are ‘‘fit for purpose’’). Currently, the only sperm counting method with a considerable body of evidence to support and clincally justify its use is the hemocytometer (Mortimer, 1994; WHO, 1999). When using the hemocytometer, the sperm number is calculated using a fixed volume of semen under the coverslip and counting the sperm in a single plane. A significant association between pregnancy and the sperm concentration measured has consistently been shown for this method (Dunphy et al, 1989; Tomlinson et al, 1996, 1999; Guzick et al, 2001). Thus, 64% of laboratories involved in the analysis of semen use this method routinely (Keel et al, 2000). However, despite its validity as a method, the use of hemocytometry is thought by many to be inconvenient, in that the hemocytometer must be cleaned and assembled prior to each counting event and it involves the use of dilution techniques that can introduce errors, either due to poor technique or the viscous nature of the semen itself. Mathematical mistakes can occur when applying the correction factor to determine the eventual counts, and the recommended dilution method uses fixatives, such as formal saline, which are often a reason for rejection of the method by embryologists in IVF laboratories. Comparisons of other counting chambers with the hemocytometer, particularly those marketed as easy-touse 1-step methods have generally not been favorable. Makler sperm counts have been shown to be generally higher than the corresponding counts obtained with the hemocytometer (Coetzee and Menkveld, 2001; Sukcharoen et al, 1994). Indeed, Ginsburg and Armant (1990) have found the Makler chamber counts to be 62% higher than those obtained with the hemocytometer when using latex beads. Other methods, such as the Leja slide (Gynotec Malden, Nieuw-Vennep, The Netherlands) or the Microcell (Conception Technologies, San Diego, Calif) have been shown to produce significantly lower average sperm counts as compared to the hemocytometer. In particular, marked differences were seen at high * Andrology Lab Corner welcomes the submission of unsolicited manuscripts, requested reviews, and articles in a debate format. Manuscripts will be reviewed and edited by the Section Editor. All submissions should be sent to the Journal of Andrology Editorial Office. Letters to the editor in response to articles as well as suggested topics for future issues are encouraged. Correspondence to: Dr Mathew Tomlinson, Fertility Unit, East Block B Floor, Queen’s Medical Centre, Derby Rd, Nottingham NG7 2UH, United Kingdom (e-mail: [email protected]). Received for publication November 16, 2006; accepted for publication January 9, 2007.