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

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 Diff-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 Diff-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 Diff-Quik stained smears was the optimal preparation method for computerised sperm morphology evaluation, comparing favourably with manual evaluations.

Slide preparation and staining procedures for reliable results using computerized morphology.

The purpose of this study was to standardize slide preparation and staining procedures to improve the efficiency and effectivity of the IVOS system on normal sperm morphology readings with regard to the strict criteria. Semen samples from patients attending the Reproductive Biology Unit, Tygerberg Hospital, were used. In experiment 1, five different Diff-Quik staining procedures, including the standard procedure, were evaluated on each of 22 patients and the effect of slide preparation within 1 h or more than 5 h after collection and the effect of immediate fixation versus fixation after 24 h were observed. In experiment 2, the manual evaluation time per slide (n = 20) by two technicians was compared with the time taken by computer. In experiment 1 the median % normal for the 5 different staining procedures was 6, 6.5, 9.5, 8.5, and 5.5%. No significant difference was found between the different staining procedures (p = .60, nonparametric Friedman test). In experiment 2 the mean time for manual assessment by two technicians was 3 min:6 s and 3 min:53 s per slide as compared to 4 min:39 s by computer. For experiment 1, slides can be prepared immediately or after 5 h. Fixation time also does not interfere with the computer's ability to identify normal forms. For experiment 2, the IVOS system is competitive regarding assessment time. Standardization of optimum staining procedures is important to ensure repeatability and comparability. Therefore, slides should be prepared immediately after liquefaction and fixed immediately after air drying.

Comparison between swim-up and glass wool column filtration of human semen in a gamete intrafallopian transfer program.

This study compared swim-up and glass wool filtration in both pregnancy outcome and fertilization of excess oocytes in patients undergoing gamete intrafallopian transfer. Gamete intrafallopian transfer patients were retrospectively included in the study group (n = 52). The criteria for inclusion were as follows: Semen had to have been prepared by means of glass wool filtration and at least 2 metaphase II oocytes had to have been transferred. Each patient from this group was then carefully matched with another patient according to specific criteria (number of metaphase II oocytes aspirated and transferred, normal sperm morphology, wife's age, the absence of anti-sperm antibodies, semen preparation by means of the swim-up procedure). Fourteen patients were matched with themselves (groups A1 and A2) and 38 patients were matched with another patient (groups B and C). The results indicate that there was no significant difference in the fertilization and pregnancy probabilities of sperm prepared by means of glass wool filtration or swim-up procedure. The comparative pregnancy rates for the groups were A1 (7.1%) versus A2 (7.1%) and B (28.9%) versus C (31.6%). Factors other than fertilization and pregnancy potential may have a greater influence on choosing the optimum sperm preparation procedure.