This document provides information about evaluating semen quality through various tests and examinations. It describes how to collect and transport semen samples, the normal characteristics of semen, and different abnormalities that can be detected through physical, microscopic, biochemical and other analyses of semen samples. The goal of semen analysis is to assess male fertility and detect any issues that may be causing infertility.

1. SEMEN ANALYSIS

2. SPERMATOLOGICAL TERMINOLOGY SPERMATOZOON = SINGLE SPERM CELL ASPERMIA = ABSENCE OFSEMEN HYPOSPERMIA = INSUFFICIENT SEMEN (<2 mL) HYPERSPERMIA = TOO MUCH SEMEN (> 6 mL) HEMOSPERMIA = BLOOD PRESENT IN THE SEMEN PYOSPERMIA = PUS PRESENT IN THE SEMEN AZOOSPERMIA = ABSENCE OF SPERMS IN SEMEN OLIGOSPERMIA = < 20 MILLION SPERMS / mL POLYSPERMIA = >250 MILLION SPERMS / mL ASTHENOSPERMIA = REDUCED MOTILITY OF SPERMS TERATOSPERMIA =>50 % ABNORMALLY FORMED SPERMS NECROSPERMIA = SPERMS PRESENT ARE DEAD CRYPTOSPERMIA = VERY FEW SPERMS, DETECTABLE AFTER SEDIMENTATION GLOBOSPERMIA = ONLY ROUND HEADED SPERMS

3. EVALUATION OF SEMEN EVALUATION OF SEMEN IS DONE : To judge the quality & quantity of semen. To know whether ejaculate is fit for extension & preservation. To know the dilution rate. To detect male infertility.

4. SEMEN Normal semen is an admixture of spermatozoa suspended in secretions (SEMINAL PLASMA ) from the glandular tissue of the male genital system. The ejaculate can be divided into four fractions: 1) PRE – EJACULATORY FRACTION : It is clear secretion of COWPER’S or LITTER’S GLANDS & contains proteins with moderately viscous consistency, which may possibly serve to neutralize residues of urine . 2)PRELIMNARY FRACTION : This originates from the prostrate gland. It gives SEMEN it’s characteristic CHESTNUT BLOSSOM ODOUR.It contain enzymes which liquefies the spermatozoal coagulum. 3)MAIN FRACTION : It originates from the SEMINAL VESICLES, TESTES, EPIDIDYMIS & partially from the prostate gland. The preliminary fraction & the main fraction contain majority of spermatozoa. 4)TERMINAL FRACTION : Is formed by secretions of seminal vesicles & is entirely gelatinous in consistency ,with large no. of immotile spermatozoa.

5. SAMPLE COLLECTION & TRANSPORT The patient should be given clear written or oral instuctions concerning the collection , handling & transport of the semen sample. The sample should be collected after minimum of 48 hours of abstinence, but not later than seven days of sexual abstinence. The ideal period of sexual abstinence is 3 – 5 days. Two samples should be collected for initial evaluation. The interval between the two collections should not be less than seven days or more than three months. Ejaculate should be collected in privacy near the laboratory. It should be delivered to the laboratory within one hour of collection. If the motility of the spermatozoa is abnormally low ,the interval between the collection & analysis of the second sample should be kept as short as possible.

6. SEMEN COLLECTION & TRANSPORT The sample is obtained by MASTURBATION in a clean wide mouthed container made of glass or plastic. The container should not be covered. It should be left open. The examination should be started after ½ hour so as to observe LIQUEFACTION. Condoms must not be used for semen collection. Coitus interruptus is not recommended as a method of collection of sample because the first portion of the ejaculate will be lost , which contains the highest concentration of spermatozoa. Collected samples should not be exposed to extremes of temperatures , i.e. less than 25 degrees or more than 40 degrees .

7. SAFETY MEASURES FOR SPERMATOLOGY LABORATORY SEMEN SAMPLES SHOULD BE HANDLED CAREFULLY. CONTACT OF SEMEN WITH OPEN WOUNDS OR SKIN SHOULD BE AVOIDED. DISPOSABLE GLOVES SHOULD BE WORN WHEN HANDLING FRESH OR FROZEN SEMEN SAMPLES. EXTERNAL SURFACE OF SEMEN CONTAINER & LABORATORY SURFACE IF CONTAMINATED, MUST BE WASHED WITH SODIUM HYPOCHLORITE SOLUTION (5.25 % ) PIPETTING BY MOUTH SHOULD NEVER BE DONE. MECHANICAL PIPETTING DEVICES MUST BE USED FOR HANDLING LIQUIDS IN THE LABORATORY. ALL PERSONNEL WORKING IN THE LAB. MUST BE VACCINATED AGAINST HEPATITIS B VIRUS

8. EVALUATION OF SEMEN SINGLE TEST CANNOT GIVE AN IDEA OF FERTILITY OF SEMEN , RATHER A COMBINATION OF 3-4 TESTS ARE REQUIRED TO REACH AT CONCLUSION. THE VARIOUS TESTS TO BE CONDUCTED ON SEMEN CAN BE CLASSIFIED AS : MACROSCOPIC/ PHYSICAL EXAMINATION MICROSCOPIC TESTS. BIOCHEMICAL TESTS. BACTERIOLOGICAL/SEROLOGICAL/IMMUNOLOGICAL TESTS.

9. GUIDELINES FOR SEMEN COLLECTION NOT RECOMMENDED COLD/SUN EXPOSED SAMPLE 10 NOT RECOMMENDED SAMPLE DURING FEVER 9 NOT RECOMMENDED ORDINARY CONDOM 8 NOT RECOMMENDED COITUS INTERRUPTUS 7 ROOM TEMPERATURE TEMPERATURE 6 CLEAN,WIDE MOUTHED GLASS /PLASTICCONTAINER CONTAINER 5 MASTURBATION METHOD OF COLLECTION 4 2 – 4 WEEKS PERIOD BETWEEN 2 SAMPLES 3 TWO IF RESULT IS ABNORMAL NO. OF SAMPLES 2 > 2 AND < 7 DAYS(3-5 DAYS) ABSINENCE 1 RECOMMENDATIONS CRITERIA

10. SPERMIOGENESIS

11. THE NORMAL SPERMATOZOON

12. PHYSICAL EXAMINATION OF SEMEN COAGULATION: Semen is ejaculated in liquid state. It gets coagulated due to enzyme PROTEIN KINASE from seminal vesicles. To observe this ,the semen sample should be collected in lab. Absence of coagulation indicates CONGENITAL ABSENCE OF VAS DEFERENS,SEMINAL VESICLES OR OBSTRUCTION OF THE EJACULATORY DUCT

13. PHYSICAL EXAMINATION OF SEMEN LIQUEFACTION AT ROOM TEMPERATURE ,NORMAL SEMEN GETS LIQUEFIED WITHIN 30 MINUTES AFTER COLLECTION. Presence of MUCOUS STREAKS indicate incomplete liquefaction. LESS THAN 20 or MORE THAN 30 MINUTES ARE ALSO INDICATIVE OF INCOMPLETE LIQUEFACTION. Sometimes the sample may not liquefy. In this situation a treatment with PLASMIN 0.35 – 0.50 UNITS/ ML or CHYMOTRYPSIN 150 USP / ML may be needed to make the sample fit for analysis. Incomplete liquefaction is indicative of dysfunctional accessory reproductive organs like prostate which leads to decreased production of prostatic enzymes.

14. PHYSICAL EXAMINATION OF SEMEN ODOUR: A normal sample has pungent odor. Absence of smell is indicative of impaired prostatic activity COLOR: Normal freshly expelled sample has a cloudy white to grey color. The degree of cloudiness depends upon sperm density. The color changes with period of abstinence. If the abstinence period is short , it is more transparent & when this period is more , it appears yellow grey in color. Inflammation of male accessory organs imparts dirty yellow color to semen ( PYOSPERMIA ). White clear semen indicates AZOOSPERMIA. Brown & red color indicate HEMOSPERMIA, OLD & FRESH BLOOD respectively.

15. PHYSICAL EXAMINATION OF SEMEN VOLUME : The volume of sample should be measured in a graduated tube or a small cylinder to the nearest 0.1 ml. Normal volume of ejaculate semen is 1.5 to 4.5 ml. If the volume of semen is below 1.5 ml , it is LOW VOLUME. WHEN SAMPLE IS BELOW 1.0 ML , SPILLAGE OR INCOMPLETE SAMPLE COLLECTION MUST BE RULED OUT. Other conditions which can lead to hypospermia or decreased volume are : 1)DISORDERS OF PROSTATE GLAND & SEMINAL VESICLES. 2)RETROGRADE EJACULATION. 3)CONGENITAL ABSENCE OF PROSTATE OR SEMINAL VESICLES. 4)OCCLUSION OF THE EJACULATORY DUCT. 5)REPEATED SAMPLE COLLECTION.

16. PHYSICAL EXAMINATION OF SEMEN VISCOSITY : This must be measured only after the semen has liquefied ,by glass rod method. A glass rod is dipped into the semen sample & raised it to measure the length of the semen thread. Normal it is 10 to 20 mm. Viscosity is more , if the length is more than 20 to 40 mm.Sometimes it is more than 80 mm. A high viscosity indicates infection in the genital tract ( prostate or seminal vesicles ) or the presence of ANTI SPERM ANTIBODIES.

17. PHYSICAL EXAMINATION OF SEMEN VISCOSITY : NEEDLE SYRINGE METHOD OF VISCOSITY MEASUREMENT : Liquefied semen sample is taken in 2 ml syringe & plunger is removed . A 20 G needle is attached to syringe & noted the time when it reaches the 1 mark & zero mark . Same procedure is repeated with water. Viscosity ratio is calculated as follows : VISCOSITY RATIO = TIME FOR SEMEN SAMPLE/ TIME FOR WATER. It should be less than 9.

18. PHYSICAL EXAMINATION OF SEMEN pH: The pH of the semen can be measured with pH paper , indicator dyes or pH mater. It depends upon sperms conc. A drop of semen is spread evenly over the pH paper & after 30 sec. the color of impregnated zone is compared to calibration strip to read the pH. The pH should be measured within an hour of ejaculation & should be in range of 7.2 – 8.0. If pH is less than 7.0 ,dysgenesis of VAS DEFERENS, SEMINAL VESICLES or EPIDIDYMIS MAY BE PRESENT . pH depends upon frequency of semen collection & quality of semen. In orchitis,seminal vesiculitis pH will be neutral or alkaline. Bacterial contaminated semen & semen with dead sperms may produce ammonia which increases the pH to make it alkaline.

19. PHYSICAL EXAMINATION OF SEMEN OSMOLALITY : Semen sample is centrifuged at very low speed ( 300 g ) & seminal plasma is separated. Osmolality of the plasma is measured in FREEZING POINT OSMOMETER after checking the Osmolality of the standard. Osmolality of seminal plasma ranges between 360 – 380 m osmol /kg . Lower Osmolality is assoc. with morphologically abnormal form ( COILED TAILS ).

20. BIOCHEMICAL ANALYSIS OF SEMEN FRUCTOLYSIS : Fructose is main sugar present in seminal plasma & is imp. nutrient for the sperms. The quality of semen can be assessed by measuring the rate of utilization of fructose. FRUCTOLYTIC INDEX is the amount of fructose used or lactic acid formed by spermatozoa per hour at 37 deg. The semen sample should be well buffered otherwise the fructolysis will stop at certain stage & result will be erroneous. FRUCTOSE CONC. = As X F X @ As = ABSORBANCE OF SAMPLE. F = MEAN FRUCTOSE STD. i.e. ½ ( 0.14 +0.28 ) @ = DILUTION OF THE SAMPLE ( it is 75 here )

21. BIOCHEMICAL ANALYSIS OF SEMEN The normal fructose value is 13 mol or more per ejaculate. Vasectomy does not interferes with secretion from the ampullae's , seminal vesicles or ejaculatory ducts & therefore fructose is also found in seminal plasma of vasectomied men . In case of azoospermia caused by congenital absence of vas deferens , low fructose level may indicate an assoc. dysgenesis of seminal vesicles. Fructose determination is also useful in rare cases of ejaculatory duct obstruction. There is positive correlation between rate of anaerobic fructolysis & deg. of motility.

22. BIOCHEMICAL ANALYSIS OF SEMEN ZINC : ZINC in human seminal plasma is 14 mg /100 ml, comes mainly from prostatic secretions . Its conc. is highest in the split ejaculate in the fraction corresponding to prostatic secretion. Like other constituents ,ZINC is unevenly distributed within prostate itself , its conc. in lateral & dorsal zones is significantly higher than in anterior portion of the gland. The conc. of ZINC & CITRIC ACID in semen gives reliable measure of prostate gland secretion. CONC. OF ZINC ( mmol / L) =( A sample/ A std. ) X 30.6 X200/1000 where 30.6 is conc. of zinc std in umol/l & 200 is dilution factor.

23. BIOCHEMICAL ANALYSIS OF SEMEN MEASUREMENT OF CITRIC ACID : Human prostatic secretion was found to be main source of citric acid . Citric acid is present in seminal plasma of mammals at widely fluctuating levels. In man ,the value is 52umol per ejaculate. There is evidence that level of citric acid in human seminal plasma reflects the human androgenic state , judging from significant correlation between blood plasma testosterone & seminal citric acid levels. CALCULATION : A1 – A2 / EXTINCTION COEFFICIENT X ABSORBANCE DILUTION =( A1- A2 /6.3 )X 58 X ( 3.02/0.2) = ( A1 – A2 ) X 139.

24. BIOCHEMICAL ANALYSIS OF SEMEN A ) HYLAURONIDASE ENZ. PRINCIPLE : Hylauronidase act on the bond bet. N-acetyl hexose amine & D glucuronate residues in hylauronic acid .When heated in alkali, the released N acetylhexoseamines form a glucorozoline intermediate , which then reacts with P dimethyl amino benzaldehyde in a acid medium to yield a blue chromophore having a max. absorbance at 585 nm. ONE UNIT OF HYLAURTONIDASE is defined as amt. of enz that causes release of 1 mmol of N acetylglucosamine per hour at 37 deg.C SIGNIFICANCE : HYLAURONIDASE HAS BEEN CONSIDERED TP PLAY AN IMP. ROLE IN CELL DISPERSAL OF CUMULUS OOPHORUS & SUBSEQUENT PENETRATION OF ZONA PELLUCIDA BY SPERMATOZOA

25. BIOCHEMICAL ANALYSIS OF SEMEN ACID PHOSPHATASE : PRINCIPLE : The method is based on enz. hydrolysis of phosphate bond of P nitro phenol phosphate .Upon the addition of base , the liberated P nitro phenol assumes a yellow color which absorbs light at 410 nm .Utilising a std curve of alkylin p nitrophenol, an estimation of enz. activity is made. P nitrophenol phosphate + h2o = p nitro phenol + h3po4. ONE INTERNATIONAL UNIT OF ENZ. ACTIVITY IS DEFINED AS THAT AMT. OF ENZ. WHICH YIELDS 1 MMOLE OF P NITRO PHENOL PER MIN.

26. MICROSCOPIC EXAMINATION OF SEMEN During the initial examination ,estimation are made for sperm density , motility , agglutination of sperms & presence of cellular elements other than sperms. A phase contrast microscope is ideal for all examinations of unstained preparations of fresh semen or washed spermatozoa. An ordinary light microscope with lowered condenser can also be used . Assessment of motility should be done at 37 deg using warmed stage. The preparation is examined at high power. If the no. of sperms per varies considerably it indicates that semen is not homogenous. In such cases the semen sample is mixed thoroughly & examined again All samples in which no sperms are detected by microscopy should be centrifuged to detect the spermatozoa in the sediment. Centrifugation at 3000 g for 15 min is recommended. When no sperms are detected after centrifugation, sample should be classified as AZOOSPERMIC.

27. MICROSCOPIC EXAMINATION OF SEMEN. Only recognizable sperms with tail should be considered in differential morphological count. Immature cells up to & included the round spermatid stage are not included as spermatozoa. Loose or free sperm heads are not counted as spermatozoa but are recorded separately. Morphological evaluation should be performed in several systematically selected areas of slide . During slide examination all normal spermatozoa are assessed & defects of abnormal spermatozoa are noted. If possible OCULAR MICROMETER can be used to estimate the size of spermatozoa.

28. MICROSCOPIC EXAMINATION OF SEMEN. SPERM DENSITY : THE NO. OF SPERMS IN 5 – 10 RANDOM HIGH POWER FIELDS ARE COUNTED & AVERAGE IS MULTIPLIED BY 1000000. This will give rough sperm count . The sample should be centrifuged if density is too low. If no sperms are observed , the sample is AZOOSPERMIC . If only 10 – 20 sperms are seen in one field , it is OLIGOSPERMIC . If the sperms are more than 30 – 40 / high power field or it is difficult to count them , then it is NORMAL DENSITY.

29. MICROSCOPIC EXAMINATION OF SEMEN CELLULAR ELEMENTS OTHER THAN SPERMATOZOA : The ejaculate invariably contains cells other than spermatozoa. These are POLYGONAL EPITHELIAL CELLS from genitourinary tract ,SPERMATOGENIC CELLS & LEUCOCYTES. Leucocytes are present in most of the human ejaculates , the predominant cell being NEUTROPHIL. Accurate assessment of no of these cells is imp. because excessive no of these cells ( LECOSPERMIA ) indicates R.T.I. Leucospermia may reduce the volume of the ejaculate , sperm concentration, sperm motility & there may be loss of sperm function. As a general rule , a normal ejaculate should not contain more than 5 x 10x6 round cells / ml , while no of leucocytes should not be more than 1 x 10x6 / ml.

30. METHODS OF DETECTING LEUCOCYTES A no of techniques has been devised for estimating the leukocyte population in the semen. Two commonly used techniques are based on presence of intracellular PEROXIDASE & LEUKOCYTE COMMON ANTIGENS. PEROXIDASE STAINING TECHNIQUE. IMMUNOCYTOCHEMISTRY

31. PEROXIDASE STAINING TECHNIQUE THIS TECHNIQUE IS BASED ON IDENTIFYING INTRACELLULAR PEROXIDASE ENZYME, A CHARACTERISTIC OF NEUTROPHILS. PEROXIDASE POSITIVE CELLS WILL STAIN BLUE WHILE PEROXIDASE NEGATIVE CELLS ARE UNSTAINED. These cells are counted in HEMOCYTOMETER for leukocytes to estimate the % age of PEROXIDASE +VE & -VE CELLS in a wet preparation.

32. IMMUNO CYTOCHEMISTRY All classes of human leukocytes express a specific antigen ( CD 45 ) that can be detected using monoclonal antibody. The cells that are not positively identified are leukocytes , spermatid , spermatocytes & spermatogonia. As only spermatozoa are included in sperm count , the conc. of other cell types can be calculated relative to known no. of spermatozoa. If N is no. of given cell type out of 100 spermatozoa counted in same fields as 100 spermatozoa & S is sperm count in millions / ml , the conc. C of the given cell type in millions/ml can be calculated using the formula : C = N x S / 100

33. MICROSCOPIC EXAMINATION OF SEMEN AMORPHOUS PARTICULATE MATTER : Semen sample may show amorphous material in background . GRADING OF AMORPHOUS MATTER IS DONE AS : GRADE 0 NO MATERIAL IS SEEN GRADE 1 MILD GRADE 2 MODERATELY SEEN GRADE 3 & 4 DENSELY PACKED WITH AMORPHOUS MATERIAL. Sample showing GRADE 3 or 4 indicates INFECTION.

34. MICROSCOPIC EXAMINATION OF SEMEN AGGLUTINATION : Agglutination means motile spermatozoa stick to one another. Adherence of immotile sperms to one another or motile sperms to mucous threads should be considered NON SPECIFIC AGGREGATION rather than AGGLUTINATION. The presence of AGGLUTINATION is suggestive of immunological cause of infertility . Agglutination should be assessed in 20 random fields & avg. %age of sperms clumped together is estimated . Type of agglutination is also noted i.e. HEAD to HEAD, TAIL to TAIL ,HEAD to TAIL & MIDPIECE to TAIL. All types of agglutinations could be due to ANTI SPERM ANTIBODIES & should be further subjected to IMMUNOLOGICAL TESTS

35. MICROSCOPIC EXAMINATION OF SEMEN MOTILITY : Spermatozoal motility is most imp. criteria of judging the fertilizing ability of semen. Percentage of active motile spermatozoa correlate well with high conception rate. It also correlates with capacitation ability of spermatozoa ,which is very imp. both in natural & in vitro fertilization. Quantitative motility is to distinguish the % age of motile from non motile spermatozoa in 10 different high power fields . Qualitative motility can be graded by two grading systems. Acco. to W.H.O. ,it has been graded under three grades : GRADE 1 = SLUGGISH GRADE 2 = MODERATELY ACTIVE GRADE 3 = VERY ACTIVE (RAPID LINEAR PROGRESSION )

36. MICROSCOPIC EXAMINATION OF SEMEN MOTILITY : SPERM MOTILITY IS GRADED ACCO. TO I.C.M.R INTO 4 GRADES GRADE I = RAPID LINEAR PROGRESSIVE GRADE II = SLUGGISH LINEAR PROGRESSIVE GRADE III = NON PROGRESSIVE GRADE IV = IMMOTILE. The duration of motility is also imp. The loss of motility after two hours should not be more than 20 % of the initial motility . Motility is estimated by mounting a drop of liquefied semen on a slide & covering it with cover slip . The slide is exam. under high power & at least 10 optical fields should be evaluated & %age of different motile & immotile spermatozoa should be calculated. RAPID LINEAR PROGRESSIVE & SLOW LINEAR PROGRESSIVE spermatozoa are counted first, followed by spermatozoa with NON PROGRESSIVE MOTILITY & IMMOTILE spermatozoa. At least 200 spermatozoa are counted.

37. MICROSCOPIC EXAMINATION OF SEMEN MOTILITY : Other systems for measurement of motility are : MULTIPLE EXPOSURE PHOTOGRAPHY using MAKLER CHAMBER. This is a micro photographic method using counting chamber. Progressive motility , Deviant motility or immotile are recorded on a photograph taken through a disc with 6 slots, driven by an intermittent electric motor, with an exposure time of 1 sec. The head of motile spermatozoa are photographed many times during the relatively long exposure time of 1 sec. , so the motile spermatozoa appear as CHAINS composed of SIX LINKS while the immotile spermatozoa are clearly visible from HEAD TO TAIL AS BRIGHTER FORMS.

38. MICROSCOPIC EXAMINATION OF SEMEN MOTILITY : LASER DROPPLER SPECTROSCOPY USING THE LAZYMOT. The LAZYMOT operate on Doppler principle & measures frequency stuff of scattered laser light caused by movements of spermatozoa in the scattered laser . Possible factors which can be investigated are : GLOBAL MOTILITY AS a %. PROPORTION OF SPERMATOZOA WITH PROGRESSIVE MOTILITY AS a %. MEAN SPEED OF SPERMATOZOA IN um/SEC. SPEED OF ACTIVELY PROGRESSIVE MOTILE SPERMATOZOA IN um/ SEC. SPEED OF POORLY PROGRESSIVE MOTILE SPERMATOZOA IN um / SEC. SPERMATOZOAL DENSITY IN MILLIONS OF SPERMATOZOA / ml EJACULATE.

39. MICROSCOPIC EXAMINATION OF SEMEN SPERM MORPHOLOGY : The assessment of sperm morphology consists of qualitative & quantitative differentiation of normal form from abnormal form. Human normal spermatozoa & its abnormal form can be identified by two methods. STAINED SPECIMEN PHASE CONTRAST METHOD ON WET PREPARATION. Different staining methods used are : PAPANICOLAOU STAINING GIEMSA STAINING BRYAN LEISHMAN STAIN SHORR STAIN

40. MICROSCOPIC EXAMINATION OF SEMEN A) PAPANICOLAOU STAINING METHOD : The pap. staining method differentiating clearly between basophilic & acidophilic cell components & allow a detailed exam. of the nuclear chromatin pattern. Sperm morphology is identified better with pap. stain with little modification as compared to routine vaginal pap stain. PRECAUTIONS : Check the acidity of the water before preparing the different grades of ethanol. The pH should be 7.0 . One dip corresponds to an immersion of about one second. Scott's solution is used when ordinary tap water is hard .

41. SMEAR PREPARATION Pipette 5 – 20 uL ( 5 uL if sperm conc. is > 20 million / ml ; 10 – 20 uL if sperm conc. is < 20 million / ml ) of well mixed semen on a clean slide . Feathering without feather can be used for all conc. of semen. Dilute & centrifuge LOW LOW Dilute & centrifuge LOW HIGH Sandwich & pull out or dilute & centrifuge HIGH HIGH Feathering without feather HIGH LOW TECHNIQUE SPERM CONC VISCOSITY < 45 DEGREES < 40 MILLION / ML > 45 DEGREES > 60 MILLION/ML 45 DEGREES 40-60 MILLION/ML ANGLE OF SECOND SLIDE SPERM CONC.

42. STAINING & EXAMINING THE SMEAR PAPANICOLAOU STAIN : Fixation req. no air dry artifacts; shows fine cellular details of all regions of the sperm ; storage stability ; used by andrology lab. recommended by WHO SHORR STAIN : give similar results to those of PAP. DIFF. QUICK STAIN : air dried ; air dry artifacts ( size of sperm head is larger than that stained by PAP or SHORR ) ; Background staining. Count 200 recognizable sperms under 100 x oil immersion. Use OCULAR MICROMETER to measure the size of the head. Pale blue Pale blue Green CD Blue or red Blue or red Blue or red Tail reddish reddish reddish Mid piece Dark blue Dark blue Dark blue Post acrosomal Pale blue Pale blue Pale blue Acrosome DIFF QUICK SHORR PAP Part of sperm

43. DEFINITION OF NORMAL SPERM < 20 % of head area Up to 4 Ambiguous 4) VACUOLES 4.5-5 u long,2.5-3.5 u wide .length/width = 1.5-1.75 3-5 u long, 2-3 u wide ( width = ½ -2/3 of length 3-5 u long, 2-3 u wide 4-5 u long, 2-3 u wide 3)SIZE 40-70 % of head surface >1/3 of head surface 40-70 % of head surface Approx.1/2 to 2/3 of head surface 2) ACROSOME Oval Oval Perfect oval, smooth border Oval HEAD 1) SHAPE WHO 3 rd WHO 2nd STRICT ASCP

44. DEFINITION OF NORMAL SPERM < 1/3 of head area < ½ of head area CD (cytoplasmic droplet 7 – 8 u LONG 7 – 8 u LONG LENGTH 1.5 X HEAD 5 u LONG 2) SIZE : LENGTH Axially attached Axially attached Axially attached Straight regular outline Straight regular outline Slender straight regular outline - MID PIECE 1) WHO 3 rd WHO 2 nd STRICT ASCP

45. DEFINITION OF NORMAL SPERM > Or =45 u long > 45 u long 10 x head 50 -55 u long 3)LENGTH Thinner than mid piece 1 u at base, 0.1 u at end 2) WIDTH Slender uncoiled ,regular outline Slender uncoiled ,regular outline Uniform size , uncoiled TAIL PIECE 1) WHO 3 rd WHO 2 nd STRICT ASCP

46. CATEGORIES DEFECTS Large -Small -Tapering -Amorphous -Double -Pyriform -Pin -Round -Vacuolated -Large -Small -Tapering -Amorphous -Double -Pyriform -Pin -Round BORDERLINE 1)NORMAL - Slight deviation from oval 2)ABNORMAL -Acrosome <40 %or >70 % of head area -Round head -Small head -Tapered head -Double head -Large head -Acrosomal abnormality -Post Acrosomal abnormality -bicephalic -Paired -Large head -Small head -Vacuolated HEAD WHO 3 rd WHO 2 nd STRICT ASCP

47. CATEGORIES DEFECT Multiple tails -Short tail -Broken tail’ -Hair pin -Coiled tail -Irregular width -Multiple tails -Short tail -Broken tail’ -Hair pin -Coiled tail -Irregular width ABNORMAL -All tail defects -Coiled tail -Curled tail -Multi tailed -Variation in length -Terminal droplet TAIL Abnormal size -Thickened -Bent tail -Missing tail -CD present -Abnormal size -Thickened -Bent tail -Missing tail -CD present BORDERLINE 1)NORMAL: -Slightly thick 2)ABNORMAL -Abnormal length -Very thick -CD present -Bent tail -Abnormal size -Thickened tail -Bent tail -Missing tail -CD present MIDPIECE WHO 3 rd WHO 2 nd STRICT ASCP

48. NORMAL SPERM

49. AMORPHOUS HEAD WIYH NORMAL MID PIECE & TAIL

50. NORMAL HEAD WITH MISSING TAIL

51. DOUBLE HEAD SPERM

52. LARGE HEAD WITH COILED TAIL

53. DOUBLE HEAD WITH COILED & DUPLICATE TAIL

54. LEFT = DEAD SPERM RIGHT = LIVE SPERM

55. AMORPHOUS HEAD WITH DUPLICATE TAIL

56. AMORPHOUS HEAD

57. AMORPHOUS HEAD WITH BENT TAIL

58. LARGE HEAD WITH COILED TAIL

59. TAPERING HEAD WITH CYTOPLASMIC DROPLET

60. SPERMIOPHAGE

61. PIN HEAD SPERM

62. SPERMATOCYTE

63. SPERMATID

64. SPERM COUNT The sperm count is no. of spermatozoa / ml. Counting of sperm count is done by many methods Most commonly used are : HAEMOCYTOMETER NEUBAUER CHAMBER THOMA – ZEISS CHAMBER PRINCIPLE: The principle is ejaculate is diluted with standard diluent's & is charged in the chamber, which is covered with cover slip & no. of spermatozoa per ml of ejaculate can be calculated. In this procedure 1 : 20 dilution is made from well mixed semen sample by diluting 20 uL semen with 0.38 ml diluent COMPOSITION OF DILUENT : SODIUM BICARBONATE 50 g FORMALIN 35 % (V/V ) 10 ml AQUEOUS GENTIAN VIOLET 5 ml

65. SPERM COUNT DETERMINATION OF SPERM COUMT After dilution ,gently mix the sample. Draw semen sample up to 0.5 mark of WBC pipette. Draw in semen diluting fluid up to 11 mark & mix well. Load the chamber & allow the sperms to settle for about 5 min. COUNT THE SPERMS IN FOUR CORNER SQUARE. CALCULATIONS : SPERMS/ML OF SEMEN = (SPERMS COUNTED IN 4 SQUARES X 10 X 20 X 100 ) / 4 NOTE : The calculation is similar to WBC formula except that reported sperm count is PER ML instead of PER CU MM .

66. SPERM COUNT 0.4 1 2 4 CONVERSION FACTORS (no. of LARGE squares counted = 4 ) DIL.SEMEN(SEMEN + DILUENT ) SPERMS PER HIGH POWER FIELD 1:50 (1 + 49 ) > 200 million 4 1:20 (1 + 19 ) 40 – 200 million 3 1:10 (1+9 ) 15 – 40 million 2 1:5 ( 1 + 4 ) < 15million 1

67. SPERM COUNT THOMA ZEISS COUNTING CHAMBER Thoma zeiss chamber has a depth of 0.1 mm & is divided by parallel lines into a grid of 16 squares. The spermatozoa are counted in five squares. The most convenient approach is to count four diagonal squares & fifth central square. Multiplying the total no of sperms from counting the five squares by 1000000 gives the no of spermatozoa per ml for a dilution of 1 : 20

68. NORMAL VALUES FOR HUMAN SEMEN 50 % OR MORE LIVE VIABILITY 7 < 1 MILLION / ML WBC’s 6 15 % OR MORE NORMAL FORMS MORPHOLOGY 5 50 % OR MORE MOTILE MOTILITY 4 4O MILLION OR MORE/ ML SPERM COUNT 3 7.2 OR MORE pH 2 2.0 ML OR MORE VOLUME 1

69. DIAGNOSTIC EVALUATION OFSPERMATOZOAL DENSITIES NO SPERMATOZOA AZOOSPERMIA AFTER SEDIMENTATION CRYPTOZOOSPERMIA 250 MILLION/ML POLYZOOSPERMIA < 20 MILLION/ML OLIGOSPERMIA 20-250 MILLION/ML NORMOSPERMIA

70. SPERM VITALITY Sperm vitality is reflection of proportion of sperms that are alive. Semen is mixed with EOSIN or EOSIN – NIGROSIN STAIN & no. of spermatozoa which are stained ( red colored ) are dead spermatozoa. At least 200 spermatozoa are counted & result is expressed in % age. In fertile subjects vitality should be more than 60 % , in sub fertile it is 40 – 60 % & value is less than 40 % in infertile males IN EOSIN –NIGROSIN, LIVE SPERMATOZOA ARE WHITE & DEAD ARE STAINED RED.THE NIGROSIN PROVIDE BLACK BACKGROUND WHICH MAKES SLIDE EASIER TO ASSESS. Sperm vitality assessment provides a check on the accuracy of motility evaluation . The presence of large no. of vital but immotile sperms may be indicative of structural defects in the flagellum.

71. SPERM FUNCTIONS TEST Routine semen analysis doesn’t give information about fertilizing ability of sperm. Therefore sperm function tests are required. Four commonly used tests are : HYPO OSMOTIC SWELLING TESTS. TEST FOR ACROSOME INTACTNESS. SPERM NUCLEAR CHROMATIN DECONDENSATION TEST. SPERM MITOCHONDRIAL ACTIVITY INDEX.

72. SPERM FUNCTIONS TESTS PRINCIPLE TEST ESTIMATES THE FULL INCIDENCE OF SPERMS HAVING FULL COMPLEMENT OF MITOCHONDRIA CONTAINING RESP. ENZYMES SPERM MITOCHONDRIAL ACTIVITY INDEX TESTING THE ABILITY OF SPERMATOZOA HEAD NUCLEUS TO DECONDENSE. SPERM NUCLEAR CHROMATIN DECONDENSATION TEST. ACROSOMAL ENZYMES DISSOLVE PROTEIN & ASSESS IT UNDER PHASE CONTRAST MICROSCOPE TEST FOR ACROSOME INTACTNESS ASSESSMENT OF MEMBRANE FUNCTION BY EXPOSING THE SPERM TO HYPO OSMOTIC SOLUTION HYPO OSMOTIC SWELLING TEST

73. SPERM FUNCTION TESTS INTERPRETATION : > 50 % S.M.A.I > 70 % DECONDENSATION TEST > 60 % ACROSOME INTACTNESS > 60 % HOS FERTILE TEST

74. IMMUNOLOGICAL TESTS ANTI SPERM ANTIBODY ( ELISA ) : PRINCIPLE : The ELISA antisperm antibody kit uses sperm antigen which are extracted from pool of treated sperms attached to wells of microlitre plates. Serial dilutions of sera are attached to the wells & after incubation , the amt. of bound antibodies is measured by appropriate antisperm ab’s ,which are covalently linked to alkaline phosphatase . This test require serum. INTERPRETATION : A POSITIVE TEST IS INDICATED BY O.D OF AT LEAST 0.3 AT 405 nm. THE DILUTION OF 1/32 OR MORE AS ANTISPERM AB’S IS POSITIVE , WHILE DILUTION OF 1/16 IS CONSIDERED BORDERLINE.

75. THANKS BY : Dr RAVI JAIN