1. Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment การประเมินวิธีการย้อมสี eosin-nigrosin แบบขั้นตอนเดียว สำหรับการประเมินค่าการอยู่รอดของตัว อสุจิมนุษย์ L.Björndahl, I.Söderlund and U.Kvist Human Reproduction Vol.18, No.4 pp. 813-816, 2003 นำเสนอโดย นางสาวอมรวดี จุ แดง รหัส 51312335 อาจารย์ที่ปรึกษา : อ. ดร. ศุภชัย ศุภ ลักษณ์นารี

3. Sperm www.rothamsted.bbsrc.ac.uk

4. Semen Stains Eosin-haematoxylin Christmas tree stain Trypan blue stain Nigrosin- Eosin stain

5. Introduction The assessment of sperm vitality is one of the basic elements of semen analysis. Motile sperm and Immotile sperm Immotile - immotile dead sperm - immotile live sperm

6. Eosin - mark dead cells Nigrosin - blackground The simplified one step technique : Add sperm into the mixture of eosin and nigrosin. Used on boar, bull and ram sperm (Campbell et al., 1956). Used on rabbit sperm (Beatty,1957) and various mammalian sperm (Dott and Foster, 1972).

7. Used on washed human sperm by Mortimer (Mortimer et al., 1990). The one - step technique is now widely in use for basic semen analysis.

8. Materials and methods Studied 1235 consecutive semen samples. The mainly men in couples consulting the clinic due to infertility problems. Sample collected by masturbation and complete vitality and motility assessments performed.

9. The one step eosin-nigrosin staining technique Contained 0.67% eosin Y and 10% nigrosin according to Mortimer (Mortimer, 1985;1994) But dissolved in 0.9% sodium chloride in distilled water.

10. 0.67 g of eosin Y yellow (CI 45380, VWR No 115935) and 0.9 g sodium chloride Dissolved in 100 ml distilled water under gentle heating Add 10 g of nigrosin (CI 50420, VWR No 115924; VWR™International)

11. Stored in a dark and sealed glass bottle Boil and allowed to cool to room temperature (20°C) Filtered through quality filter paper (Munktell, quality 3: 90 g x m -2 retention of coarse and gelatinous precipitates; filtration speed 700 ml x min -1 x 100 cm -2 ; VWR No 108016-9; VWR™ International)

12. Method Semen samples were liquefied at 37°C for 30 min One droplet of semen + One droplet of the eosin- nigrosin staining solution Incubated for 30 s at room temperature (20°C ) 12  l droplet was transferred to a labelled microscope slide and smeared

13. The smears were air dried and examined directly At least 200 sperm were assessed at a magnification of 1000 x under oil immersion with a high-resolution 100 x bright field objective www.vivo.colostate.edu

14. White sperm = Live sperm Red sperm or pink sperm = Dead sperm Slight pink or red appearance restricted to the neck region (leaky necks) = Live sperm Reliability and repeatability of assessment : IQC (inter-individual variation was <10%) Exposure time to eosin-nigrosin was varied from 30-300 s.

15. Motility assessment Assess at least 400 sperm in each semen sample. Four motility categories. – Rapid progressive – Slow progressive – Non-progressive – Immotile

16. Using techniques for assessment and quality control according to the World Health Organization(WHO) The percentages of motile sperm (the sum of the three categories of motile sperm)

17. Calculation and evaluation The sum of percentages motile and stained (dead) sperm in a sample should be <100% ( some immotile but live sperm) If assessed without errors the sum of percentages of motile and stained(dead) sperm cannot be >100% The sum of dead and motile sperm is >100% (overestimation of the percentage of motile sperm, or to an error in the technique for vitality assessment rendering too high a percentage of stained (dead) sperm)

18. results

19. Technique 1 =1235 sample Technique 2-4 = 20 sample The mean for sum of stained (dead) and motile sperm using the one-step eosin- nigrosin technique was 91%(SD + 10%)

20. Figure 1. Proportion stained + motile sperm (mean, SD) distributed into classes according to percentage of stained (dead) sperm. (A)The present study (n = 1235). (B) Graph drawn from data in Eliasson and Treichl (Eliasson and Treichl, 1971), (n =724).

21. 1 A :The distribution of sums for percentage stained and percentage motile sperm was similar regardless as to whether the samples have many or few dead sperm. 1B : the original evaluation of the eosin alone method (Eliasson and Treichl, 1971)

22. The percentage of live sperm did not change with increasing time of incubation (30-300 s) in the eosin-nigrosin staining solution Mean percentages of live sperm were 81, 80, 81 and 80% for incubation times of 30, 60, 180 and 300 s respectively

23. Discussion the sum of percentages for dead and live sperm is 100%. Live sperm are either motile or immotile. the sum of percentages for dead and motile sperm should be somewhat <100%.

24. The sum of percentages for motile and stained (dead) sperm: technique 1 - mean 91% (95%CI:72-110) (the present 0.67% eosin-nigrosin technique) technique 2 - mean 96% (95%CI:72-120) (the 0.5%-eosin-alone technique) technique 3 - mean 95% (95%CI:74-116) (the two-step 1%-eosin and nigrosin technique) technique 4 - mean 109% (95%CI:87-131) (the two-step 5% eosin-nigrosin technique)

25. The present technique gave the narrowest 95% confidence interval. The wider total range in the present results (45-120%) : n=1235 Technique 4 showed a systematic error (sum> 100%)

26. The increased proportion of dead sperm cause: - Toxic effect of the 5% eosin concentration. - Stored of stained. - Unmounted smear in humid environment. - Made and dry smear. The one-step eosin-nigrosin technique minimizes the exposure of sperm to eosin (concentration and duration of exposure)

27. 30 s for mixing and incubation. Environmental temperature,mixing and incubation, volume used for making the smears and the size of the slides were kept constant 30-300s in nigrosin mixture before smear : do not affect the final percentage of dead sperm in human semen.

28. Comparison between the one step eosin alone technique with the one-step eosin- nigrosin technique : sum of percentage motile and stained lower with few dead sperm. Run at 37  C, QC and training of technicians.

29. Change solvent, specific volume and technique smear : the Nordic Association for Andrology and the Special Interest Group in Andrology of the European Society of Human Reproduction and Embryology

30. conclusion Modified from Mortimer. Evaluated with sperm motility data : WHO Reliability in assessing sperm vitality in samples. Using standard bright-field microscope. Few methodology steps to control management. Recommended in the basic semen analysis when sperm vitality is to be assessed.

31. Thank you