SEMEN ANALYSIS, physical chemical and microscopy

semen analysis

1. SEMEN ANALYSIS MITHUN VENUGOPAL HOD, ASST. PROFESSOR DEPT. OF PATHOLOGY

2. INTRODUCTION Problems with male semen account for 40% of all infertility. Semen analysis is the best way to test male infertility. It gives important information about the quality and quantity of the sperm.

3. Production and Composition Semen consists of four fractions contributed by the testis , bulbourethral gland , seminal vesicle and the prostate . Spermatozoa or sperms are produced in the testis and mature in the epididymis, where they are stored until ejaculation. Sperms account for about 1-2% of the total volume of ejaculate. The secretory product of the bulbourethral gland functions to lubricate the urethra.

4. Production and Composition The majority of the semen is contributed by the seminal vesicle in the form of an alkaline viscous coagulum which contains prostaglandins and fructose. The prostatic fluid in semen is acidic, contain acid phosphatase and proteolytic enzymes which acts on the coagulum and helps in liquefaction of the semen.

5. Indications Evaluation of infertility. To evaluate the effectiveness of surgical therapy. To select appropriate donor for therapeutic insemination. Screening for exposure to reproductive toxins. To evaluate the effectiveness of vasectomy. In suspected cases of rape, paternity issues and other medico legal cases.

6. Patient instructions Patient should be given clear and simple instructions explaining the need for semen analysis and what is required for specimen collection. Patient should be informed about the importance of abstinence time. Ejaculate must be collected after 3-5 days (but not more than 7 days) of abstinence. Prior to sample collection, the patient must void and wash hands and genitals to minimize the chances of contamination. Samples should be obtained by masturbation and collected in a sterile, nontoxic plastic or glass wide-mouth container. All sample containers are labelled with adequate information to eliminate any chances of error.

7. Patient instructions Ideally, the samples must be collected close to the laboratory. If the specimen cannot be produced close to the laboratory, it must be delivered to the laboratory as soon as possible, certainly within 1 hour of collection. During this period, the sample has to be kept warm under the clothing and temperature extremes must be avoided. The WHO recommends obtaining two samples for initial evaluation at an interval of not less than 7 days or more than 3 weeks. If the results from the two samples are distinctly different, additional samples have to be collected and examined.

8. Information on the sample container and patient sheet Once the patient has collected the specimen, some preliminary information about the specimen should be obtained. Patient’s complete name & Hospital number. Collection date and time. Abstinence time in days. Time the sample is received at the laboratory.

9. Physical examination LIQUEFACTION TIME: A normal sample usually liquefies within 60 minutes at room temperature although usually this occurs within 15-20 minutes. It is determined by the time required for the gelatinous mass to liquify. A normal sample might contain gel-like gelatinous corpuscles that do not liquefy. Exact liquefaction time is of no diagnostic importance unless >2 hours elapse without any change. This may indicate poor prostatic secretion since the liquefying enzymes are derived from the prostate gland. On the other hand, absence of coagulation may indicate ejaculatory duct obstruction or congenital absence of seminal vesicles.

10. Physical examination VOLUME: Volume of the ejaculate should be measured by transferring the liquefied sample into a graduated 15 ml conical centrifuge tube. The normal volume of ejaculate after 2-5 days of sexual abstinence is about 2-6 ml. Retrograde ejaculation, obstruction of lower urinary tract (urethra, congenital absence of vas deferens, seminal vesicles) may yield low volume.

11. Physical examination According to the WHO, the reference value for semen volume is ≥ 2.0ml; however, for clinical purposes; semen volume is differentiated into three categories to facilitate interpretation and diagnosis: Aspermia: No semen produced after orgasm. Hypospermia : <0.5 ml of semen ejaculated (partial or complete retrograde flow of semen, accessory glands impairment). Hyperspermia : > 6 ml of semen ejaculated (long period of sexual abstinence or overproduction of fluids from the accessory sex glands). If the volume is <1 ml it is important to determine if the sample is complete. The highest sperm concentration is seen in the initial ejaculate.

12. Physical examination COLOUR AND APPEARANCE: Normal – homogenously opaque, whitish grey or pearly white. Yellow – pyospermia . Blood tinged – bleeding in the seminal vesicle.

13. Physical examination VISCOSITY: Viscosity measures the seminal fluid’s resistance to flowing. It is measured by the length of the ‘thread-lines.’ It can be estimated by using a glass rod and observing the length of thread that forms on withdrawal and using a Pasteur pipette, expelling the semen drop by drop. A normal sample leaves small, discrete drops; abnormal samples will form threads more than 2 cm long. High viscosity may interfere with determinations of sperm motility. Viscosity can be categorized as normal, moderate or high.

14. Physical examination PH: Normal semen pH is in the range of 7.2 - 8.2, and it does tend to increase with time after ejaculation. Any change in the normal range of pH may be caused by inflammation of the prostate or seminal vesicles. The pH of liquefied semen is determined by using pH test strips.

15. Microscopic examination MOTILITY OF THE SPERM: Place a small drop of semen on a glass slide and cover it with a coverslip. Examine under high power objective under reduced light. Count atleast 200 sperms and grade the motility of each of the sperm. Sperm motility is the ratio of the number of motile sperm to total number of sperm in a given volume and is expressed as a percentage.

16. Microscopic examination Grade – 0: No motility Grade – 1: Slow forward progression and mild lateral movement. Grade – 2: Slow forward progression with substantial yaw. Grade – 3: Slightly faster forward progression with little yaw. Grade – 4: Spermatozoa moving rapidly in a straight line with little yaw and no lateral movement.

17. Microscopic examination The number of sperms belonging to each grade is expressed in percentage. Normally the individual should have >25% of sperms belonging to grade 3 or 4 and >50% of sperms in grade 2, 3 or 4. The sperm motility is recorded at the end of 2, 3, 6 hours. The number of motile sperms decreases by 5% per hour after four hour of collection. Necrozoospermia : Total absence of motility. Asthenozoospermia : Decreased sperm motility .

18. Microscopic examination SPERM COUNT: Mix the specimen after liquefaction. Draw the semen up to 0.5 in a WBC pipette. Draw the semen diluting fluid (sodium bicarbonate, formalin, distilled water) up to mark 11 and mix well. Charge the improved Neubauer counting chamber and allow the sperm to settle for 5 minute. Count all 4 squares of WBC area. Normal: 40-300 millions/ml.

19. Microscopic examination Oligozoospermia : Sperm count <20 million/ml. Azoospermia: Absence of sperms. Causes of low sperm count: Infections (mumps, orchitis, prostatitis, etc.) Obstruction, Endocrinopathies

20. Microscopic examination SPERM MORPHOLOGY: After liquefaction make a thin smear on a glass slide. Allow the smear to dry and then heat fix. If necessary remove the mucous by dipping the slide in semen diluting fluid and then into buffered distilled water. Stain the smear using Leishman’s stain.

21. Microscopic examination MORPHOLOGY OF NORMAL SPERM: It has a head which measures 4um in length and 3um in diameter. It has a cap called acrosomal cap which stain slight blue in color. The nucleus of the sperm stains dark blue. The middle piece measures 7um in length and 1um in diameter. The neck measures 0.3um which is situated between the head and the middle piece. The tail measures 45-50um in length and stains red or pink.

22. Microscopic examination ABNORMAL SPERM FORMS: Morphological abnormalities can be found in head, body or tail. Abnormality of the head: Too large (macrocephaly) or too small (microcephaly) head, double heads, pointed heads, ragged heads, vacuoles in the chromatin, etc. Abnormality of the middle piece: Absence of middle piece, bifurcated or swollen middle piece. If the middle piece defects are >25% it is pathological. Abnormalities of the tail: double, triple, quadruple tails, rudimentary or absence of tail, cytoplasmic vacuole along the tail indicate an immature sperm. If the defects are >25% it is pathological.

23. Microscopic examination

24. Microscopic examination SPERM VIABILITY TESTING: Assessing the ability of the sperm plasma membrane to exclude extracellular substances. The cytologically intact ‘live’ cells can be determined using several vital staining techniques such as eosin Y and trypan blue.

25. Microscopic examination EOSIN-NIGROSIN STAIN: An Eosin- Nigrosin stain must be done on all specimens having a motility of 30% or less. The stain must be performed immediately following the initial motility examination. Eosin- Nigrosin stains the dead sperms and live sperms are unstained. Add 2 drops of 1% aqueous eosin Y and 3 drops of 10% aqueous Nigrosin . Mix well. Immediately make two thin smears from this mixture and air dry. Count 100 sperm on each slide in duplicate using high power (×40). Calculate percentage of viable (unstained) and non-viable (stained) sperm.

26. Chemical examination QUANTITATION OF SEMEN FRUCTOSE: Fructose reacts with resorcinol in a strong acidic medium to give a red colored complex. Normal value: 150-300 mg/dl High fructose level is associated with low sperm count and vice versa. Low fructose levels are associated with decreased testosterone.

27. Microbiological examination ORGANISMS FOUND IN SEMEN Ureaplasma Mycoplasma Chlamydia trachomatis Neisseria gonorrhoeae Trachomonas vaginalis E-Coli HIV HPV CMV