View stunning SlideShares in full-screen with the new iOS app!Introducing SlideShare for AndroidExplore all your favorite topics in the SlideShare appGet the SlideShare app to Save for Later — even offline
View stunning SlideShares in full-screen with the new Android app!View stunning SlideShares in full-screen with the new iOS app!
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
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
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.
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
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.
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 :
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.
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.
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
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
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
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
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
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
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
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.
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