Effects of different staining and washing procedures on the results of human sperm morphology evaluation by manual and computerised methods

Abstract

The purpose of this study was to investigate the effect of different staining and washing procedures on the results of human sperm morphology evaluation by manual and computerised methods. Furthermore, it was intended to find the staining and washing combination which would provide optimal readability for computer-assisted sperm morphology evaluations. In phase one, four staining methods were evaluated for smears prepared from the resulting samples following a two times washing procedure. In phase two, 20 semen samples were used to compare the DS-Quik and Papanicolaou staining methods, following one and two washes. All manual readings, of Papanicolaou and Diff-Quik stained smears, were comparable with each other, with means between 7.3% and 7.9% normal spermatozoa. All the manual readings were also comparable to the computer readings of the DS-Quik slides following one and two washes with means of 9.0% and 5.9%, respectively. However, due to the higher computer readings found for the Papanicolaou stained smears, with means of 13.9% and 13.5% following one and two washes, respectively, a statistically significantly difference between overall computer and manual readings was found (Wilks' Lamda, P= 0.0002). Taking all data into consideration, it could be concluded that the one wash DZ-Quik stained smears was the optimal preparation method for computerised sperm morphology evaluation, comparing favourably with manual evaluations.Correspondence: R. Menkveld PhD, Andrology Laboratory E3, Tygerberg Hospital, 7505 Tygerberg, South Africa.

Assessment of Spermatozoa Morphology under Light Microscopy with Different Histologic Stains and Comparison of Morphometric Measurements

“…Menkveld et al (1997) examined the relationship between human spermatozoa morphology and function of acrosome. Acrosome reaction evaluated at normal, small and large size in living and dead spermatozoa with triple stain.…”

Comparison of three staining methods for the morphological evaluation of human spermatozoa

“…In all these reports, the Diff-Quik â stain was found to be a suitable method, especially for the computer-assisted sperm morphology analysis (20), because the computer appears to recognize Diff-Quik â -stained sperm better than using other techniques. However, a direct comparison of the Diff-Quik method with the other stains appears to result in higher percentages of morphologically normal spermatozoa (17,31). Coetzee et al (32) found a significant bias of 1.6% with Diff-Quik â versus the Papanicolaou stain in favor of higher percentages of morphologically normal sperm using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS; Hamilton Thorne, Beverly, MA).…”

“…Because of the wet mount, spermatozoa could be lying on their sides rendering confusion with tapering forms by inexperienced observers (36). Another reason could be that the staining procedure itself is causing the sperm to swell, for which there is some indication (17). Other disadvantages are that these slides cannot be stored (36,37), as well as their high costs (33).…”

“…On the basis of these results, the 1992 WHO manual (16) introduced the Diff-Quik â method together with a number of other staining techniques as an alternative to the Papanicolaou technique. Since then the Diff-Quik â staining technique has been compared repeatedly with other staining techniques as reference for the purpose of a more rapid staining procedure or for obtaining optimal contrast for the computer-assisted sperm morphology evaluation in both human and animal semen samples (17)(18)(19)(20)(21)(22). Another rapid sperm staining method, introduced by Schirren et al (23), is the stain-coated Testsimplets â slides, which frequently are used mainly in smaller centers and practices for assisted reproduction because of time constraints.…”

The usefulness of Real Time Morphology software in semen assessment of teratozoospermic boars

“…The ability of spermatozoa to bind to the zona pellucida, initiates the acrosomal reaction and binding to the oolemma that are necessary for a normal fertilization [Marnet et al 2000]. These phenomena occur in a subpopulation of spermatozoa with normal morphology [Menkveld et al 1997]. Moreover, Ron-el et al [1991] showed that human spermatozoal morphological defects are correlated with reduced male fertility, a lowered fertilization rate, poor embryo morphology, and reduced pregnancy rate.…”