Semen examination

Semen Examination , Speical and routine tests by suchit kumar

1. Semen examination special & routine test Moderated by:- Presented by:- Dr. S.P. Khanna Suchit Kumar (Assoc. Prof. ) Msc(mlt) 1 st year Department of pathology ( MMIMSR ) M.M. Deemed to be university

2. introduction Semen (seminal fluid ) consists of spermatozoa(sperms) and fluid part. About 40% cases of infertility are due to abnormalities in semen. Semen analysis is the first test to be performed while investigating for infertility. Defect of sperms may be quantitative (absence f sperms , lacks of enough sperms) and qualitative.

3. Indications of semen analysis Assessment of fertility/ infertility. Success of vasectomy. Success of varicocele surgery ( after 3-4 months of surgery) Medicolegal purpose: In alleged rape cases, vaginal pool smear are examined to detect sperms. For selection of assisted reproductive technology (e.g. in vitro fertilization, gamete intra-fallopian transfer technique).

4. Different procedures of semen analysis Sample collection Physical examination Microscopic examination Chemical examination Immunological assays Microbiological assays Sperm function tests Semen cryopreservation Sperm donation

5. Collection of the semen sample Patient is asked to collect the semen by masturbation after a minimum of 2 days and a maximum of 7 days of sexual abstinence. Specimen should be collected in a clean,dry,wide mouthed plastic/glass container. Collection of condom sample is not advisable. The sample submitted in the laboratory immediately within one hour of collection for examination.

6. Physical examination Semen is examined grossly for the following features: colour ,volume,viscosity,pH reaction and liquefaction. Colour normally , volume of semen is b/w 2.5 to 5 ml. Viscosity when ejaculated , semen is fairly viscid and it falls drop by drop. PH reaction normally, it is slightly alkaline with pH b/w 7-8. Liquefaction occurs because of presence of fibrinolysis. Normally liquefaction occurs at room temperature within 10-30 minutes.

7. Microscopic examination Semen is examined microscopically for motility, count and morphology.

8. Motility Place a drop of liquefied semen on clean glass slide. Put a coverslip over it and examined it under the microscope . Normally , within 2 hours of ejaculation , more than 60% of spermatozoa are vigorously motile and in 6-8 hours 25-40% are still motile is. If motility is less than 50%, a stain for viability such as methylene blue or eosin Y with nigrosin as countersatin can be done. Heads of non-motile sperms are stained positive for the dye. Sterile moles have <25% motility of sperms.

9. count This is done in Neubauer’s (hemacytometer) chamber using a WBC pipette. Draw liquefied semen in WBC pipette up to marks 1 and then the diluting fluid up to mark 11. The composition of diluting fluid is as under: Sodium bicarbonate 5 gm Formalin (neutral) 1 ml Distilled water 100 ml continued….

10. count After mixing it properly, charge the chamber. Allow the spermatozoa to settle down in 2 minutes. Examine under microscope and count the number of spermatozoa in one large peripheral square. Calculations: number of sperms= n In 1 µl , no. of sperms = n X 10 no. of sperms per µl = n X 10 X 10 no. of sperms per ml = n X 10 X10 X1000 n X 100,000 ( 1 lakh) Average no. of sperms counted in 1 mm² Normal value = > 60 million/ml Abnormal value = < 20 million/ml

11. morphology Prepare a thin smear from liquefied semen on a glass slide and stain with a Romanowsky stains , Pap or H&E stain. Observe at least 200 sperms for any abnormality in their morphology Normally 80% of sperm are normal The abnormal forms of sperms are with double head , swollen and pointed head , double tail and rudimentary forms.

12. morphology of sperm

13. chemical examination Chemical analysis of semen consist of two main tests : Fructose test Acid phosphatase test

14. fructose test This test determines androgen deficiency or ejaculatory obstruction of semen ; the level of seminal fructose is low in both these conditions. Normal seminal fructose level is 150-600mg/dl Fructose is measured by qualitatively by resorcinol test.

15. Acid phosphatase test The test is used for seminal stain and on veginal aspirate in medicolegal cases Normally , semen has 2500 KA units /ml of acid phosphatase

16. Immunological assays The presence of sperm antibody binding to head or tail antigen considered specific for immunologic infertility. The antibodies are usually of immunoglobulin A (IgA) or IgG, and rarely of IgM class. These are detected by direct or indirect mixed agglutination test .

17. Microbiological assays Genital track infections by bacteria , yeast and sexually transmitted diseases may have significant adverse effect on male infertility. If the concentration of bacteria exceed 1000 CFUs per ml , The colonies should be identified and test for antibiotic sensitivity .

18. sperm function test Defective sperm function may affect various fertilizing functions . Most importantly , it includes transport of sperms in the male and female reproductive tracts , which is responsible for fertilization activity such as specific zona binding , penetration and formation of male pronucleus . continued…

19. Sperm function test A list of common sperms function test is as under : Sperm penetration assay to test the success of penetration of egg by the spermatozoa. Hypoosmatic swelling test to test the membrane integrity of the sperms. Cervical mucus penetration test to test the relative ability of motile sperms to pass through the cervical mucus of the partner collected at mid-cycle.

20. Semen cryopreservation Cryopreservation or semen banking is indicated in the following conditions : For assisted reproduction For donor insemination For men undergoing vasectomy In men before starting cancer therapy In life-threatening jobs (e.g. military service )

21. Sperms donation Sperm donation is a procedure in which a man donates semen — the fluid containing sperm that is released during ejaculation — to help an individual or a couple conceive a baby. Donated sperm can be injected into a woman's reproductive organs (intrauterine insemination) or used to fertilize mature eggs in a lab (in vitro fertilization). The use of donated sperm is known as third-party reproduction.