1 Semen analyses and Leja ® Comparison of different counting methods

2 Correct counting of semen There is a relationship between number of cells and human fertility. There is a relation between preferred method of treatment and number of cells: –Waiting –IUI, IVF, ICSI Amount = (better choice of) Quality

3 Important aspects of semen analysis Semen is a suspension Semen need to be diluted Precision: –Reproducibility of obtained results Accuracy: –Correctness of results = correlation to golden standard –Measurements taken by calibrated instrument

4 Fertility of a semen sample Sperm count Motility –Hyper activation Morphology Stability of fertility when diluted and in uterine tube

5 Counting What do we want to know? –Number of normal cells –Number of moving cells –Number of white blood cells

6 Methods of counting Haemocytometer FACS Coulter Counter Corning SP 100 CASA system with Leja ® slides

7 Haemocytometer I

8 Haemocytometer II Golden standard Time consuming Error-sensitive: precision in making dilutions 2 x 200 cells to count Statistical error of counting alone ± 25% No differentiation between dead/alive; motile/non-motile cells

9 Haemocytometer III In conclusion, the haemocytometer is only usable to calibrate other counting systems.

10 FACS Measures the fluorescence of individual cells Uses an internal standard Measures 2000 – 5000 cells per measurement Cells remain alive (X – Y separation) The fluorochrome needs to be specific and are limited to 5 different Staining of nucleus, dead/alive staining, DNA lesions, proteins, receptors

11 Coulter Counter I

12 Coulter Counter II Counting large numbers is possible: smaller counting errors Differentiation between particle size is possible Differentiation between different cell types is not possible Suitable for pure suspensions

13 Corning Measuring the optical density One is not sure about what exactly is being measured Very high error sensitivity

14 SP 100 (Nucleocounter) I

15 SP 100 II Measures haploid cells (= semen) using quantitative fluorescence microscopy Effect of laminar flow (SS-effect) not researched Measures many cells, relative low error. Special cassettes are needed

16 Accuracy All systems have been calibrated to haemocytometer Each manufacturer is obliged to show calibration data Conclusion 1: All systems count cells, but not motility

17 CASA systems Measures concentration Measures motility Uses disposable counting chambers i.e. Leja ® slides Fluorescence microscopy: –FACS possibility at a fraction of the costs

18 Conclusions so far InstrumentFACSImproved Neubauer CorningSperm Vision UltimateSP 100 CV2.72 %7.14 %10.4 %8.13 %5.40 %3.11 % 95 % confidence interval 2.45 – – – – – – 3.49 Comparison of FACSCount AF System, Improved Neubauer hemacytometer, Corning 254 photometer, Sperm Vision CASA System, Hamilton Thorne Ultimate CASA System and NucleoCounter SP-100 for determination of sperm concentration of boar semen C. Hansen a, *, T. Vermeiden b, J. Vermeiden, C. Simmet, B.C. Day, H. Feitsma b a The National Committee for Pig Production, Danish Bacon and Meat Council, Axeltorv 3, DK-1609 Copenhagen V, Denmark

19 Leja I Leja is the company that develops and produces in favour of assisted reproduction Leja’s ideas originate in an academic setting and are translated to products that work in everyday practice

20 Leja II Leja is ISO 9001:2000 certified All Leja slides are CE-marked

21 Leja slides I Two sheets of glass cured together at fixed distance –Using non-toxic resin –resin holds spacers of known defined diameter –Only sharpened glass is completely flat Very small variations in chamber heights occur

22 Leja slides II The glass surface is treated: –Cleaning –Coating No direct contact between sperm cell and glass Regular microscopic glass is surface active: –Sperm cells bind to clean glass –Residues of soap / oil are toxic to sperm –Quality is not consistent glass directly from regular stock is not suitable to determine motility!

23 Leja slides III

24 Leja quality control Semi-automated production leaves room for both human error and permanent quality control: –Printing errors –Chamber height –toxicity

25 Slide production I Production facilities in clean room environment Screen printing of ink and glue layers

26 Slide production II Curing cover slip by UV induction

27 Newton bands Using monochromatic light, Newton bands are an indication of chamber height

28 R&D quality control Random testing of chamber heights by special interference meter (only 3 on earth) Toxicity testing: <7% less motility after 6 minutes

29 Quality certificate Each order of Leja ® slides comes with a quality certificate (available to download on

30 Leja contact details Leja Products B.V.