Semen analysis by Dr.Renukadevi

IMA

1. SEMEN ANALYSIS DR.RENUKADEVI SENIOR EMBRYOLOGIST ARC International Fertility & Research Centre

2. Introduction A semen analysis measures the amount of semen a man produces and determines the number and quality of sperm in the semen sample. A semen analysis is usually on e of the first test done to help determine whether a man has problem fathering a child (infertility) A problem with the s emen or sperm affects more than one- third of the couples who are unable to have children (infertile)

3. Semen Analysis SA are mainly carried out to determine whether:- A man has a reproductive problem that is causing infertility. A vasectomy or vasectomy reversal has been successful SA is the singular test for fertility in male that can provide information on sperm production. patency of the male ducts. the function of the accessory glands. and ejaculative function . Since semen samples may vary from day to day, 2 or 3 samples may be evaluated within a 3-6 month period for accurate testing. A semen analysis to test the effectiveness of a vasectomy is usually done 6 weeks after the vasectomy

4. Semen collection Masturbation, directing the semen into a clean sample cup. Do not use a lubricant which can kill sperms Coitus interruptus - withdrawing the penis from the partner just before ejaculating follow by ejaculating into a clean sample cup. Coitus - by using a condom. A special (silicon) condom that does not contain any substance that kills sperm ( spermicide ). After ejaculation, carefully remove the condom from the penis. Tie a knot in the open end of the condom and place it in a container that can be sealed in case the condom leaks or breaks. Ordinary condoms should not be used since they usually contain spermicides Assisted ejaculation – electro-ejaculation used in paralegics

5. Once after collecting the semen sample from the patient, receiving time is noted down. The sample has to be liquefied for 20-30mins at 37°c by placing the sample in heating block. (Avoid crystallization by leaving the sample stationary for a too long time). After 20-30mins of liquefaction, mix the sample homogeneously by using transfer pipette. Sample volume, viscosity and pH should be noted. SEMEN ANALYSIS

6. Semen Collection Abstinence days 2-5 Pass urine Wash hands with soap, dry Collect the entire sample into the wide mouth sterile container, 70% of sperms is in the first part of the ejaculate Keep the sample at body temperature, no sunlight Deliver the sample within one hour of ejaculation Good and reliable SA results starts from semen collection, preferably by masturbation

7. Parameters (WHO criteria 2010) Parameters agreed on consensus Parameter Lower Reference Limit Semen volume (ml) 1.5 Sperm concentration (10 6 /ml) 15 Total sperm number (10 6 /ejaculate) 39 Progressive motility (PR, %) 32 Total motility (PR +NP, %) 40 Vitality (live sperms, %) 58 Sperm morphology (NF, %) 4 pH* >/=7.2 Leucocyte* (10 6 /ml) <1 MAR/ Immunobead test* (%) <50

8. Terminologies of Semen Analysis Oligospermia – sperm concentration <15 million/ml Asthenozoospermia – <40% grade (PR+NP) or < 32 PR% Teratozoospermia – <4% spermatozoa OAT = Oligo-astheno-teratozoospermia Azoospermia – no spermatozoa in semen Polyzoospermia – ++ high sperm concentration, >200M/ml Hypospermia – semen volume < 1.5 ml Hyperspermia – semen volume > 4.5 m l Aspermia – no semen volume Pyospermia – leukocytes present in semen, >1M/ml Hematospermia – red blood cell present in semen Necrozoospermia – “dead” sperm

9. Macroscopic Examination WHO criteria 2010 Description Appearance Normal: Whitish to grey opalescent Yellow (urine, jaundice); Pink/Reddish/Brown (RBCs) Liquefaction Normal: 15 –30 minutes after collection Lumpy >60 min – sperms may be trapped in unliquefied jelly; maybe sign of prostatic infection, lack of prostatic protease Viscosity Normal Smooth and watery Abnormal –, thick with long threads (21G needle). High viscosity impede sperm movements MIX SEMEN THOROUGHLY BEFORE ANALYSIS –USE A PIPETTE

10. Macroscopic Examination Volume Normal: 1.5 ml per ejaculation Low volume (<1ml) reflect a problem with the seminal vesicles and prostate – a block, retrograde ejaculation, infection or lack of androgen. Low semen volume cannot neutralize vaginal acidity High semen volume dilute sperms/ active infection pH Normal: =/>7.2 (alkaline) Acidic pH (<7.0) in a low volume & density sample indicates –congenital bilateral absence of vas deferens (in which seminal vesicles are also poorly developed) and ejaculatory duct obstruction. pH increases with time as natural buffering capacity of semen decreases – therefore high ph is not clinically useful

11. A microscope objective of 20x should be used for counting. Place a drop of the sample into Makler chamber and count the sperm heads as follows: 10 squares of the field. This number represents the concentration of spermatozoa in millions per milliliter. When counting a sample of oligozoospermia (less than 15million/ml) all 100 squares have to be counted. Adding "00000" to that number, you will get the concentration of the spermatozoa per milliliter. SPERM COUNT Microscopic Examination

12. Grade A (fast progressive) sperms are those which swim forward fast in a straight line - like guided missiles. Grade B (slow progressive) sperms swim forward, but either in a curved or crooked line, or slowly (slow linear or non linear motility). Grade C (non-progressive) sperms move their tails, but do not move forward (local motility only). Grade D (immotile ) sperms do not move at all. Immotile spermatozoa are to be counted in a defined number of squares. Afterwards the motile spermatozoa are counted and classified into four categories (from A "fast progressive" to D "no motility"). This procedure is to be repeated in different areas of the grid to have the quality and motility to be calculated on base of this data in percent. Motility is graded from A to D,

13. SPERM MORPHOLOGY A drop of sample is placed in the sterile glass slide and covered using a cover slip, name and SID number should be mentioned. The sample is viewed under light microscope with a magnification of 40x. A Normal S perm has:    A smooth, oval-shaped head that is 5-6 micrometers long and 2.5-3.5 micrometers wide (less than the size of a needle point)  A well-defined cap ( acrosome ) that covers 40% to 60% of the sperm head  No visible abnormality of neck, mid-piece, or tail  No fluid droplets in the sperm head that are bigger than one half of the sperm head size.

14. The following categories of defects should be noted. Head defects, namely large, small, tapered, pyriform , globe, amorphous heads, vacuolated heads, double heads and small acrosome . Neck and mid-piece defects namely bent neck, abnormal mid-piece (thin or thick). Tail defects namely short, multiple, hairpin, broken, bent tails, coiled tail. Multiple defects Agglutination Pus cells/HPF Debris After completion of analysis the results should be noted in the record and typed in the patient report entry column.

15. Morphology assessment Sperms must be stained for an accurate assessment Strict criteria /Kruger / Tygerberg The sperm head is oval, smooth-symmetrical outline, a length of 3-5 um and a width of 2-3um. The head should have a well defined acrosome area of 40-70% and vacuoles (=/<2) that occupies ~ 20% head area. The mid-piece must be straight and slender, 0.5 um in width and 7-8um long, straightly aligned to the head. The tail must be straight and 45-50 um long. To be classified as normal, the sperm must be normal in all portions (head, mid-piece, tail). At least 400 sperms must be scored on randomly chosen fields. Normal Forms (%) = normal sperms / the total number of sperms evaluated x 100 .

16. Diff-Quick Staining Simple, quick staining method. Moderate differentiation of structures --the acrosome is stained red and the post acrosomal area dark red. Very thin smears are necessary as the background is influenced by the presence of protein in the seminal fluid and tends to be dark. The slides are fixed with fixative ( Cytospray , Kinetik , Australia). Diff Quik 1 for 5-6 seconds. Clean water x 2. Removed excess moisture. Diff Quik 2 for 10 seconds 2-3 dips in clean water. It is left to dry by standing the slide on one end on absorbent paper towel. Sperms stained by Diff- Quik are larger by comparison to H&E staining as the cells did not undergo dehydration by alcohol.

17. Abnormal Sperms

18. Abnormal Sperms Triple head sperm Acrosome reacted sperm Sperm with no acrosome Sperm with a tapering head and swollen mid-piece

19. Morphology Assessment Assessment is subjective due to the wide variation in sperm sizes and shapes. % NF correlated well with the fertilization rate in-vitro and pregnancy rate [Kruger et al, 1996; Morgenthaler et al, 1995] Sperms with defective heads are more likely to be immotile than sperms without defects, and defective motile sperms tend to show sluggish motility as compared to normal sperms [ Aitken et al, 1995].

20. Vitality Assessment Eosin- nigrosin (dead sperm stain pink/red) Eosin (1%) (dead sperm stain pink/red) Trypan (0.4%) blue (dead sperm stain blue) Hypo-osmotic swelling test (HOS) (live sperm shows tail curling Test 1, 2 and 3 for diagnostic uses. Usually 1:1 ratio of semen to dye mixture, mix well and smear onto a slide. Read immediately at x40 objective, count 200 sperms Test 4 is use to choose live (immotile) sperm for ICSI Dead sperms will not react in HOS while live sperm will take up fluids causing their tails to curl within 5 min and stabilize at 30 min. Therefore viable sperms may be selected for ICSI [Lin et al, 1998; Cayan et al, 2001].

21. Vitality Assessment Dead sperms are stained pink/red Live sperms shows curling tails