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Dr. Varad Vardhan Bisen

Product of: Testes- constitutes 2-5% of volume. Epididymis –stored in epididymis,

maturation occurs, pottasium,sodium and glycerylphosphorylcholine secreted by epididymis.

Vas Deferens-sperms travel through vas to the ampulla which serves as storage area.Ampulla secretes ergothioneine and fructose(source of nutrition).

Seminal vesicles (50%) Flavin (fluoresce) Fructose(viability + motility)

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Potassium Ascorbic acid Prostaglandins.

Prostate (40%) – milky Acid phosphatase Coagulation + Liquefaction enzymes Citric acid

Bulbo-Urethral glands-secrete mucus.

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Assessment of fertility-first step in investigation of infertility.

Forensic purposes Effectiveness of vasectomy - 2 samples 1

month apart , confirming the absence of sperms.

Suitability for artificial insemination Paternity testing For selection of assisted reproductive

technology.4

Macroscopic viscosity coagulation +

liquefaction volume pH

Microscopic Concentration motility morphology viability

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Motility &Viability must be performed within 1½ - 2 hrs of collection

REMEMBER

SEMEN IS A BODY FLUID

BIOHAZARDOUS

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Name –label the sample. Period of abstinence – 3 days.If period of

abstinence is less than 3days, sperm count is lower.

Time of collection + analysis recorded Entire ejaculate and not coitus interruptus

in a wide mouth container.Specimen should be kept close to the body temperature.

Condom collection not recommended due to spermicidal agents.

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Semen can be directly taken from epididymis by PESA(per cutaneous epididymal aspiration).

Delivered within 1 hour of collection Avoid temperature extremes Two specimens should be examined-2-3

weeks apart.

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If results of 2-3 assessments differ greatly, additional samples must be analyzed.

If a sperm function test is to be performed, the sperm must be separated from fluid within 1 hour of ejaculation.

For vasectomy evaluation, only the presence of sperm, viable or nonviable is enough.

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Count >15 million/ml(WHO in 2010),older definitons state> 20 million/ml

Volume 2.0-6.0 ml

pH- 7.2-8.0

Total count -By WHO, lower reference

limit(2.5th percentile) is 39 million per

ejaculate. Morphology- >30% of normal morphology. Viability-> 75% of sperm cells alive.

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WBC< 1million/ml Motility within 1 hour of ejaculation:-

Class A->25% Rapidly progressive. Class A and B- >50%

Progressive(less linear in progression)

MAR TEST- <50%motile sperms with adherent particles.

Immunobead test-<50% motile sperms with adherent particles.

RBC none.11

Aspermia: No semen ejaculated Hematospermia: Blood present in semen Leucocytospermia: White blood cells present in

semen Azoospermia: No spermatozoa found in semen Normozoospermia: Normal semen parameters Oligozoospermia: Low sperm concentration Asthenozoospermia: Poor motility and/or

forward progression Teratozoospermia: Reduced percentage of

morphologicaly normal sperm Necrozoospermia: No live sperm in semen Globozoospermia: Round headed acrosome-

less sperm 12

(1) Appearance :

(a) Colour

1. Normal- greyish white to pale yellow.

2.Pyospermia- Turbid

3. Haematospermia- Rusty red

4. Jaundice- yellow

(b) Consistency: 13

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1.Slightly viscid &

opaque

Normal

2. Watery -Old sample

-Retrograde ejaculation

3.Thick,lumpy -Low hydration

-Frequent ejaculation.

(2) Volume :-

1.Normal - 2-5 ml

2.Hypospermia- < 1.5 ml

3.Hypersperrmia- > 5 ml

4.Aspermia- no sperm.

(3) Odour : Acrid musty odour

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Coagulation of semen: fibrinogen

Liquefaction : proteases, pepsinogen,

amylase, hyaluronidase, transaminase.

alpha-amylase used to liquefy semen for

Artificial insemination.

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Total Fructose. Total Zinc. Total acid

phosphatase. Total citric acid α-glucosidase. Carnitine

>13 µmol/ejaculate. >2.4 µmol/ejaculate. >200U/ ejaculate.

> 52 µmol/ejaculate. >20 mU/ejaculate. 0.8-2.9

µmol/ejaculate.

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pH- Measured by pH paper and pH is recorded after 30 seconds.

Normal pH is 7.2 – 8.0 after 1 hour of ejaculation.

Acidic pH- stored specimen. contamination by urine. chronic inflammation of

seminal vesicle.

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(2) Fructose : Normal : 150 - 300 mg / dl Low fructose : blockage of ejaculatory duct Absence of fructose : Agenesis of seminal vesicles Measured by : seliwanoff’s test Seliwanoff reagent – 50 mg resorcinol 33 mg conc. HCl 100 ml distilled water

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(2) Fructose : 5 ml resorcinol reagent + 0.5ml

semen mix & boil for 20-30 minutes red precipitate – fructose present

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Absence of fructose indicates obstruction

proximal to seminal vesicles or lack of

seminal vesicles.In a case of azoospermia, if

fructose is absent, it is due to the

obstruction of ejaculatory ducts or absence

of vas deferens, and if present,azoospermia

is due to failure of testes to produce sperm.

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Neutral α-glucosidase originates solely from the epididymis and its measurement is of diagnostic value for distal ductal obstruction when considered with hormonal and testicular findings.

Acid phosphatase is secreted by prostate-seminal fluid that is more alkaline than normal(pH>8) and has reduced acid phosphatase suggests prostate dysfunction.

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Wet mount: approximate count sperm agglutination pattern viability motility morphology

Preparation: mix drop on glass slide and apply coverslip

Important: volume (10ul) of semen and the dimensions of the coverslip (22x22) be standard so that we have fixed depth (20um).

Observe 10-20 using 40x-60x If number vary from field to field; not mixed

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Normally seen: Mature cells make up the greatest

percentage of cells Epithelial cells of genital tract: many in

urethritis Immature germ cells WBC

Abnormally seen: Gross bacteria Trichomonas Candida

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Sperm motility is essential for penetration of cervical mucus,traveling through the fallopian tube and penetrating the ovum.

Principle- All motile and non-motile sperms are counted in randomly chosen fields in wet preparation under 40x objective.

Method- A drop of semen is placed on a glass slide,covered with a coverslip that is ringed with a petroleum jelly to prevent dehydration, and examined under 40 x objective. Atleast 200 sperms to be counted in different fields.

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Result of motility is expressed as a percentage- A) Rapidly progressive spermatozoa. B) Slowly progressive spermatozoa-crooked or

curved movement. C) Non-progressive-movements of tail,but with

no forward progress. D) Immotile spermatozoa. Normally >25% of sperms show rapid

progressive motility or >50% of sperms show rapid progressive and slow progressive motility.

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Sperm Viability or Vitality- Principle- A cell with intact cell membrane

will not take up the eosin Y and will not be stained, while a non –viable or dead cell will have damaged cell membrane,will take up the dye, and will be stained pink-red.

Method- Mix one drop of semen with 1 drop of

eosin-nigrosin solution and incubate for 30 seconds.

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The smear is made from a drop placed on a glass slide.

The smear is air dried and examined under oil immersion. White Sperms are classified as live or non viable and red sperms are classsified dead or non viable. Minimum 200 spermatozoa to be examined.

Result is expressed as a proportion of viable sperms against non-viable integer percentage.75% or more sperms are normally live or viable.

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Staining techniques used are papanicolou, eosin- nigrosin , hematoxylin- eosin and rose bengal toluidine blue stain.

Atleast 200 spermatozoa should be

counted under oil immersion.

Head , neck, tail to be examined.

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Head is pear-shaped. Most of the head is occupied by condensed chromatin.Anterior 2/3rd is surrounded by acrosomal cap. Acrosomal cap is a flattened membrane bound vesicle containing glycoproteins and enzymes.

Neck- short segment connects head and tail. Centriole in the neck give rise to axoneme of the flagellum.

Tail- consists of –a) middle piece. b) principal piece. c) End piece.

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Normally > 30% of spermatozoa should show normal morphology.

Defects in morphology associated with infertility in males includes:-

A) Defective mid-piece - reduced motility B) Incomeplete or absent acrosome -

inabiltiy to penetrate the ovum. C) Giant head- defective DNA

condensation.

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Abnormal morphology-WHO morphological classification of human spermatozoa-

1. Normal sperm 2. Defects in head- a) large heads. b) small heads. c) tapered heads. d) pyriform heads. e) round heads. f) amorphous heads. g) Vacoulated heads. h) double heads.

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Slight flattening of the right postacrosomal area

Tail implanted slightly to the right

Left side spermatozoon: Head = Abnormal due to elongated posterior of postacrosomal region.

Right side spermatozoon: Head = Normal. Although the right side of the postacrosomal area is slightly flattened this is not enough to classify the spermatozoon as abnormal.

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Absence of the acrosome and pointed anterior head segment

Very small acrosomal area

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Small size and absence of acrosome with typical round-head defect or globozoospermia

Moderate to severe elongation of the postacrosomal area

3) defects in neck- Bent neck and tail forming an angle >90º to the long axis of head.

4) Defects in middle piece: a) asymmetric

insertion of midpiece into head. b) Thick or

irregular midpiece. c) Abnormally

thin midpiece.42

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5) Defects in tail: a) Bent tails. b) short tails. c) Coiled tails. d) irregular tails. e) Multiple tails. f) tails with irregular

width. 6) cytoplasmic droplets- >1/3rd the size of

the sperm head. 44

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Manual methods Hemocytometer or counting chamber

Improved Neubauer Hemocytometer method Makler counting chamber(no dilution) Horwell fertility counting chamber

Computer assisted Oligospermia<20 million If azospermia: fructose level must be

ordered to verify the integrity of the vas and seminal vesicles

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1. Thoroughly mix specimen and dilute 1:20 with diluent. (To obtain this dilution, dilute 50 uL of liquefied semen with 950 uL of diluent)

2. Thoroughly mix diluted specimen and allow a drop (10 - 20 pL) to into each side of the hemocytometer covered with a coverglass.

3. Allow chamber to stand for about 5 minutes in a humid container to prevent drying. During this period, the cells settle and can be more easily counted.

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Preparing appropriate diluent (distilled water + NaHCO3 + formalin +

gentian violet or trypan blue)

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4. After cells have settled, place chamber under phase contrast microscope (preferably), using 40x

5. Count spermatozoa present in 5 1/25 mm squares in center square millimeter on both sides of the hemocytometer. Each side should be tallied separately. If there is more than 10% variation between the two sides, another chamber must be prepared and the count redone. Only morphologically mature germinal cells with tails are counted.

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1 - It should be determined using the haemocytometer method- 10 ul of the thoroughly mixed diluted specimen

is transferred to the counting chamber - Counting spermatozoa in the central square of

the grid (25 large squares ) , the number of large squares are determined based on the number

of spermatozoa per each square . - Calculate total number by using the formula.

2 – alternative method : - 1 ml well-mixed sample + 19 ml cold

water - Fill a hematology counting chamber - Count number of sperm seen in a 1 mm2

area ( 16 small squares ) . This number is then multiplied by 200,000 ) “ Double figure and add 5 zeros ”!!

Decreased: vasectomy (should be 0 after 3-6 months) varicocele primary testicular failure (Klinefelters) secondary testicular failure congenital vas obstruction retrograde ejaculation endocrine causes (prolactinemia, low

testosterone)

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Immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.

The antibodies can be tested in the serum , seminal fluid or cervical mucus.

Antibodies bound to the head of the sperm-prevent the penetration of the egg by the sperm.

Antibodies to the tail-retard motility.

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A) spermMAR test: This test can detect IgG and IgA antibodies against sperm surface in semen sample.

a) directspermMAR IgGtest-drop of each semen(fresh and unwashed),IgG-coated latex particles and anti-human immunoglobulin are mixed together.

If spermatozoa have antibodies on their surface,antihuman immunoglobulin will bind IgG-coated latex particles to IgG on the surface spermatozoa, motile,swimming sperms with attached particles will be seen.

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If the spermatozoa do not have antibodies on their surface, they will be seen swimming without attached particles;the latex particles will show clumping due to binding of their IgG to antihuman immunoglobulin.

In direct spermMAR test IgA test-drop of each fresh unwashed semen and of IgA-coated latex particles are mixed on a glass slide.The latex particles will bind to spermatozoa if spermatozoa are coated with IgA antibodies.

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Indirect spermMAR test- Fluid without

spermatozoa is tested for the presence of

antisperm antibodies.Antibodies are bound to

donor spermatozoa which are then mixed with

the fluid to be analysed. Antibodies are then

detected as direct tests. If >50% of

spermatozoa show attached latex

particles,immunological problem is likely.

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2) Immunobead test- Antibodies bound to

the surface of the spermatozoa can be

detected by antibodies attached to

immunobeads. Percentage of motile

spermatozoa with attached two or more

immunobeads are counted amongst 200

motile spermatozoa.>50%spermatozoa with

attached beads is abnormal.59

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These test are available only in specialized

andrology labs.These tests are not

standardized thus making interpretation

difficult.If used singly,a sperm function may

not be helpful.They are more predictive if

used in combination.61

1) Postcoital test-The test is supposed to examine

interaction between sperm and mucus of the

cervix. Cervical mucus is aspirated with a syringe

shortly before the expected time of ovulation and

2-12 hrs after intercourse. Gross and microscopic

examination is carried out to assess the quality

of cervical mucus and to evaluate the number

and motility of sperms. If >10 motile sperms are

observed the test is considered normal.

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2) Hamster egg penetration assay-sperm are incubated with several hamster eggs that have had the outer membranes removed. After seven to twenty hours, the number of sperm penetrations per egg is measured.

3) Hypo-osmotic swelling of flagella- functional integrity of the plasma membrane by observing curling of flagella in hypo-osmotic conditions.

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4) Computer assisted semen analysis (CASA)-A catch-all phrase for automatic or semi-automatic semen analysis techniques. Most systems are based on image analysis, but alternative methods exist such as tracking cell movement on a digitizing tablet.Computer-assisted techniques are most-often used for the assessment of sperm concentration and mobility characteristics, such as velocity and linear velocity.

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Semen culture-the semen sample is tested

for the presence of bacteria, and , if present,

their sensitivity to antibiotics is determined.

Interpreting this test can also be

problematic! It is normal to find some

bacteria in normal semen samples. Culture

is done in Gram positive and Gram negative

Media.66

THANK YOU

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