Sperm Function Tests

1. Sperm Function Tests ShahinGhazali PhD student Yazd Reproductive Sciences Institute

2. Introduction: • During recent years, apart from the light microscopical determination of sperm count and morphological malformation, evaluation of function of sperm parameters has become a powerful tool in andrological laboratories.

3. These assays determine: • Biochemical parameters: a-glucosidase Polymorphonuclear granulocyte(PMN)-elastase • Biological functions: Motolity Membrane integrity Morphology Zona binding Acrosome reaction Acrosin activity Oolemma binding Chromatin condensation DNA integrity

5. Sperm function test were developed to detect abnormalities: - In sperm survival - Transport in the female genital tract - Different steps of fertilization

6. Types: • Vitality tests • Sperm-mucus Interaction Tests • Capacitation • Acrosome Reaction • Zona Binding assays • Hamster Ovum Penetration test

7. Vitality tests

8. Vitality tests: • Are used when: Low percentage motility Lost their flagellation because of metabolic dysfunction or axonemal defects Necrozoospermia(dead sperm)

9. Vitality tests: • Hypo-osmotic swelling test (HOS) • Eosin test • Eosin-nigrosin

10. Hypo-osmotic swelling test (HOS) • Simple test for integrity of plasma membrane • Using hypo-osmotic solution • Influx of water • Introduced by Jeyendran et al. (1984) Result: Intact sperms: swelling of the tail into various sizes and shape Dead cells: normal tail shape because of leaky membrane

14. Eosin Test: • Based on the fact that eosin is excluded by live cells. • Can be used in ICSI • Result: • Damaged cells: are stained specifically

15. Sperm-mucus Interaction Tests

16. Sperm-mucus Interaction Tests • In vivo postcoital test • In vitro sperm-mucus interaction test • In vitro sperm cervical mucus contact test • In vitro slide test • In vitro capillary test (Kremer test)

17. Cervical mucus: • First barrier for sperm migration • Highly viscous entire menstrual cycle • Penetrable only for a few preovulatory days • Mucus quality is very important for sperm assessment.

18. In vivo postcoital test • 9-24 h after coitus • Examining both penetration and survival of sperm • Using high magnification microscope • Result: • Presence of motile sperm= +

19. In vitrosperm-mucus interaction test • Abstinence for 3 days • Aspiration from endocervical canal • Administrating ethinyl estradiol for insufficient volume • Evaluation by WHO scoring system: Volume Viscosity Ferning Spinnbarkeit (elasticity) Cellularity ( No. of leukocytes or other cells) pH

20. In vitro sperm cervical mucus contact test: • Detecting antibodies on sperm or in mucus • Performing immediately after semen liquifaction • Mixing one drop of semen and mucus on a slide • Result: • Shaking sperm >25% = +

21. In vitro slide test • A mucus drop in the center of slide • Semen seep from the edge of cover slip • 30 min incubation • Result: • Normal semen: fingerlike penetration> 90 %

22. In vitro capillary test (Kremer test) • Mucus loaded into a flat capillary tube (Sealed one end) • The open end put vertically into reservoir of semen • Incubation for 1 h at 37c • Scoring vanguard sperm at 1, 4, and 7 cm • Result: • Excellent to poor penetration or negative penetration

23. Penetrak Test • Commercial • Using bovine cervical mucus as a surrogate in the penetration test.

24. Androlog Mail • The bovine mucus penetration test does measure important characteristics of sperm function. It measures • 1. the strength and effectiveness of sperm motility and • 2. the surface characteristics of sperm that allow them to interact appropriately with the fluids overlying the mucosal surfaces of the female tract. • However, clinically speaking, the BMPT does not add that much information. If mucus penetration is a problem for a patient's sperm, then chances are good that other semen characteristics will also be deficient, and IUI, which bypasses the cervical mucus, or another ART will be prescribed. So clinically, it may not add much to our standard Andrology tests. • I am still using the BMPT if physicians order it specifically, which has become rather rare. • I order the Penetrak Kits from Bio-Chem Immunosystems, 100 Cascade Dr., Allenton, PA 18001, 1-800-345-3127 (product #4913000). I last ordered them in September, 1998. • Erma Z. Drobnis, Ph.D. Clinical Assistant Professor Obstetrics & Gynecology and Surgery/Urology Director, Fertility Laboratories University of Missouri-Columbia 3211 S. Providence Dr. Columbia, MO 65201 U.S.A. Tel: (573) 882-7176 Fax: (573) 884=4492 drobnise@health.missouri.edu

25. A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test • D. Mortimer1, S.T. Mortimer, M.A. Shu and R. Swart Endocrine/Infertility Clinic, Department of Ob/Gyn, University of Calgary, Health Sciences Centre 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada • Correspondence: 1To whom correspondence should be addressed • Fiffy-one comparisons were made of human sperm migration into capillary tubes containing either human cervical mucus (‘Kremer test’) or a synthetic mucus substitute consisting of a 5 mg/ml solution of sodium hyaluronate (average mol. wt 2 x 106) in a phosphate-buffered medium. • The results of these two tests were highly significantly correlated and dependent upon the same sperm characteristics reflecting sperm progressive ability (including the specific movement characteristic of lateral head displacement amplitude), morphological normality and cellular vitality as well as the concentration of these more functional cells in the semen. • The result of the hyaluronate migration test, in conjunction with the mucus quality measures of Insler score and pH, allowed a 92.2% correct prediction of the Kremer test outcome (90.9% of normal tests and 93.1% of abnormal tests). In this data set, these values also corresponded to the sensitivity and specificity of the analysis, respectively. From these studies, we propose the hyaluronate migration test as a useful adjunct to routine semen analysis, sperm movement analysis and the more traditional in-vitro tests of sperm-cervical mucus interaction in the diagnostic investigation of infertile couples. It effectively assesses the mucus-penetrating potential of a semen sample without the need for relatively large quantities of midcycle cervical mucus; it will therefore augment (as an internal control), although not necessarily replace, the homologous Kremer test and reduce the quantity of both patient and donor mucus needed for comprehensive crossedhostility format testing of sperm — mucus interaction

26. Capacitation

27. Capacitation • Cell surface changes • Hyperactivated motility • Vigorous nonprogresive motion with forceful amplitude bending

28. 1 • Sperm wash • Incubation in albumin-containing culture • Very simple • No need for oocyte or mucus 2 • Using CASA 3 • Chlortetracycline-staining • Detection by fluorescence microscopy • Acrosome-reacted sperm show a staining patterndifferent from that of capacitated sperm with acrosomes still intact

29. 2 • Using CASA: • Distinguish hyperactivated from nonhyperactivated sperm, mainly by: the high curvilinear velocity low linearity large value of the amplitude of lateral head displacement of the former

31. Computer-aided sperm analysis

32. CASA: • Several comercial manufactures provide CASA system: Hamilton Thorne Hosbon sperm Tracker • Parameter settings: The user responsible for cheking and set up Use video tapes as quality control

33. Preparation of samples: • Maintain at 37c • Standard concentration is 25-50* 10 6 • Using Dulbecco’s PBS-glucose-BSA medium

34. Pic page 106

35. CASA terminology: • VCL  =  curvilinear velocity(micron/s) Time-average velocity of a sperm head along its actual curvilinear path, as perceived in two dimensions in the microscope. • VSL =  straight line velocity  (micron/s) Time-average velocity of a sperm head along the straight line between its first detected position and its last. • VAP  =  average path velocity (micron/s) Time-average velocity of a sperm head along its average path. This path is computed by smoothing the actual path. • BCF = beat cross frequency (beats/s) The average rate at which the sperm's curvilinear path crosses its average path. • LIN = linearityThe linearity of a curvilinear path. • STRstraightnessThe linearity of the average path.

36. medeaLAB CASAA new system for objective semen analysis which offers:Focus on routine requirementsReal-time processing for simultaneous assessment of up to 200 moving objectsSperm tracking for the duration of several secondsAttractive price as well as reliable support and after-sales service. Key features: • Automated analysis of sperm motility and morphology. Calculation of a complete spermiogram in under 30 seconds, multiple recordings for each sample are possible • Storage of patient data and sample recorded on template in MS Access™ and SQL-Server • Classification of sperm motility according to WHO categories • Print of spermiogram by means of user-defined HTML templates • Automatic export of data into MS Excel™ for further statistical analysis • Storage of images from morphological analysis • Full RecDate database integration (Germany only) • Use of standard hardware reduces investment need

40. MAIN SOFTWARE FEATURES: • Acquisition of images and clips in AVI format from an imaging device. • Storage of textual data, images and clips in the built-in MultiMedia Catalog database. You can take the advantage of all the powerful features of MultiMedia Catalog in application to semen analysis. The database is a perfect tool for archiving of patient data, analysis results, video clips and images, fast and flexible search and spectacular reporting with images, graphs and other features adjusted to your particular needs. Herewith our database can serve the needs of virtually any laboratory or research institution. • Automated analysis of sperm concentration and motility on native samples according to WHO recommendations. • Automatedmorphology analysis on stained samples according to strict Krueger’s criteria. • Manual assessment of concentration of white blood cells, immature germ cells, round cells. • Manual measurements for special research or other individual tasks.

44. Computer assisted semen analyzers in andrology research and veterinary practice. • Verstegen J, Iguer-Ouada M, Onclin K. • University of Liège, Department of Animal Clinical Sciences, Small Animal Reproduction Bd Colonster 20, B44, B 4000 Liège Belgium. • Abstract • The evaluation of sperm cell motility and morphology is an essential parameter in the examination of sperm quality and in the establishment of correlations between sperm quality and fertility. Computer-assisted sperm analysis (CASA) allows an objective assessment of different cell characteristics: motion, velocity, and morphology. The development and problems related to this technology are raised in this review, paying particular attention to the biases and standardization requirements absolutely needed to obtain useful results. Although some interesting results, mainly in humans, have already been obtained, many questions remain, which have to be answered to allow for further development of this technology in veterinary medicine, clinical fertility settings, physiological, and toxicology research activities. The main problem is related to the standardization and optimization of the equipment and procedures. The different CASA instruments have all demonstrated high levels of precision and reliabilityusing different sperm classification methodology. Their availability gives us a great tool to objectively compare sperm motility and morphology and to improve our knowledge and ability to manipulate spermatozoa.

45. Acrosome Reaction

47. Tests for acrosome reaction: • Triple stain • Trypan blue • Fluoroscent lectins and antibodies

48. Triple stain: • The sperm suspensions from the swim-up were diluted with an equal volume of the defined medium containing 0.2% trypan blue, incubated at 37؛C for 15 minutes, smeared on prewarmed glass slides, and air dried. The slides were rinsed in water and blotted. The smears on slides were fixed in 3% glutaraldehyde solution in 0.2M phosphate buffer at room temperature for 45 minutes, rinsed in water, and air dried. The smears were stained in 0.5% Bismark Brown solution in 30% ethanol at 40؛C for 10 minutes, rinsed briefly in water, and air dried. Finally, the smears were stained with Rose Bengalto evaluate acrosomal status, following distinction of live cells from dead ones usingtrypan blue. After staining, the slides were examined at 1,000x under phase-contrast microscopy and spermatozoa were classified into the following four categories17: • 1. live spermatozoa with acrosome reaction - light rose postacrosomal regions and white "acrosomal regions"; • 2. dead spermatozoa with normal acrosomes - blue postacrosomal regions; • 3. dead spermatozoa with abnormal acrosomes (i.e.; degenerative acrosome reactions) - blue postacrosomal regions with white "acrosomal regions"; • 4. live spermatozoa with normal acrosomes - light rose postacrosomal regions and pink acrosomes.

50. 1. a live spermatozoon acrosome reacted; 2. a dead spermatozoon with an intact acrosome;  3. a dead spermatozoon with degenerative acrosome reaction;  4. live spermatozoa with intact acrosomes;  5 and 6. live spermatozoa with acrosome partially intact.