Semen Practical Biochem

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Presented by: Yasmine Amr

Assistant lecturer in MedicalBiochemistry department


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Female sex hormones

Progesterone (21 C)

It is secreted from:

Corpus luteum. Placenta (after 10 weeks of pregnancy).

It is also formed in the adrenal cortex as a

precursor of C19 and C21corticosteroids

hormones .


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Estrogens (18 C): They are secreted from

mature graffian follicle inovaries.

They contain 18C, the first

ring is completely unsaturated

(aromatic) with no CH3 groupat C10.

There are 3 types: Estrone

(E1), Estradiol (E2) and Estriol

(E3)

The most circulating one is E1

while the most active one is E

2


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Male sex hormones (Androgens)19C

Testosterone is the most potent one. It is

synthesized in the testis.


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Diagnosis of pregnancy


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Pregnancy can be diagnosed bymeasuring Human chorionicgonadotropin(HCG) in blood or urine

Detectable amounts ~ 5 IU/L

Appears 8-11 days after conception andreach the peak(~ 100,000 IU/L) at 8-10 weekspregnancy.

In case oftwin pregnancy, the amount of HCG

is doubled.


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Serial HCG can be used to determine

abortion and ectopic pregnancy: In normal pregnancy, HCG doubles in 1.5

days in the first 5 weeks then every 2-3 days

after 5 months In ectopic pregnancy or abortion HCG rises

more slowly or even decreases


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Diagnosis of fetal anomalies

Down syndrome:is usually due to anextra copy of chromosome 21. It iscommonly associated with increased

maternal age. Neural tube defects:e.g. anencephaly,

meningomyocele,encephalocele. Usuallyassociated with folic acid deficiency duringpregnancy


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These two serious anomalies can be

diagnosed in second trimester (between

16-18 weeks) using triplet test,Measuring:

-fetoprotein (FP),

unconjugated estriol 3,

and HCG.

Down syndrome: FP, 3, HCG Neural tube defects: FP, 3, HCG


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Infertility

Infertility is defined as inability of a couple

to conceive after at least 1 year of

unprotected, well timed intercourse. This

may be due to male, female or acombination of both causes.


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Laboratory tests to determinefemale infertility:

Blood tests that measure the levels ofvarious

hormones aid greatly in determining the cause of

infertility. Some examples include:

Luteinizing hormone (LH) Follicle-stimulating hormone (FSH)

Prolactin (PRL)

Estradiol Progesterone


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Because changes in pituitary or thyroid

function can also affect the menstrual

cycle and ovulation, blood tests that

measure thyroid function(TSH and/or T4) and steroids, such

as testosterone and DHEA-S (dehydroepiandrosterone sulfate is used in

producing androgens and estrogens), are

also informative.


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Laboratory tests to determinemale infertility:

Those include blood tests and more

important semen analysis

Blood samples can be used to measure:

Free and total testosterone

Luteinizing hormone (LH)

Follicle-stimulating hormone (FSH)

Prolactin (PRL)


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Semen analysis

Semen is made up of the secretion ofall the

accessory glands of the male genital tract:

Testes5%

Seminal vesicle46-48% Prostate13-33%

Bulbourethral gland2-5%.

Semen is a grey opalescent fluid which is

formed at ejaculation. It is composed of

suspension ofspermatozoa in seminal plasma.


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Semen analysis

Physical properties

Volume

Colour

pH

VicositySpecific gravity

Biochemical tests

Fructose

Acid phosphatase

ASA

Acrosin

Zinc

L-carnitine

Alpha glucosidase

Microscopic examination

count

Morphology

Motility

ViabilityNon sperm cells


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PHYSICAL PROPERTIES OFSEMEN

Volume: Average volume is from 2-5 ml/ejaculation. Abnormalities:

1. Aspermia: Total absence of ejaculation (rare).2. Hypospermia: the seminal fluid volume is lessthan 2 ml.

3. Hyperspermia: Increased volume of semenabove 10 ml (rare).


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Colour Greyish white. It is opalescent due to its high

content of protein and the presence of morethan 60 million sperms /ml.

abnormalities:1. Urine produces pale yellow discoloration easily

detected by the consistency of the semen andthe urineferous odor.

2. Jaundice: bilirubin will also cause coloration ofsemen in deep jaundice. The semen may be avery bright yellow.

3. Blood (haematospermia) traces of fresh bloodwill color semen pink, while large amounts ofblood give bright red color.


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pH:

Between 7.3-8.1 only recorded on freshsemen by using pH paper with a range of7-9.

Inflammatory conditions of the prostate or

seminal vesicle may alter the pH of semen.Viscosity: Normal viscosity is that which allows

semen to be poured drop by drop out of

the container. It is measured the timetaken by one drop to leave the standardpipette.

Specific gravity: 1.028


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MICROSCOPICALEXAMINATION:

This includes:

Sperm

count

motility

morphology

Non-sperm cells


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Sperm count

Total sperm count is the number of

sperms in an ejaculation.

Normally, it is 20 million/ml, i.e. about 60

millions/ejaculation.

It is obtained by multiplying the sperm

concentration by the volume.


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How to conduct a sperm count

Hemacytometers were developed forcounting blood cells, but can also be usedto count spermatozoa. A hemacytometer

has two chambers and each chamber hasa microscopic grid etched on the glasssurface. The chambers are overlaid with aglass coverslip that rests on pillars exactly

0.1 mm above the chamber floor. Thus,the volume of fluid above each square ofthe grid is known with precision.


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Procedure

The semen must be killed to prevent movement

and diluted before loading into the

hemacytometer. This can be done by diluting thesemen into a buffer containing a small quantity

of formaldehyde. The dilution factor must be

recorded to allow calculating the concentration.

When there are 20-25 cells per large square, thesample is at the proper dilution.


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The example at right shows

red lines where cells on the

line would be counted. If red

dots represent cells, one

would count 3 cells in the top

middle large square.


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At least two chambers should be counted, including

at least 100 cells within each central counting areaof each chamber.


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count/ml = (Dilution Factor)(Count in 5squares)(0.05 X 106)

By convention, sperm concentration is

usually expressed in terms of sperm X

106/ml.


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Abnormalities:

1. Azoospermia means no spermatocytes(male sterility).

2. Oligozoospermia mean less than 20million/ml less than 50 millions/ejaculation

3. Polyzoospermia may reach 350millions/ejaculation


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Motility:

Percentage motility (the percentage ofsperms in the seminal fluid which arehighly active) is performed soon after theproduction of the sample and is repeated

after 1,2,3 and 6 hours after semenproduction.

Normally, after one hourthere must be

over80% active sperms.


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W.H.O divided grades of motility into:

ARapid forward progress motility

BSlow or sluggish progressive motility

CNon progressive motility

DImmotility. The cutoff value for normal is 50% grade A+B or

25% grade A motility.

Asthenospermia:sperm motility less than theWHO cutoff levels


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Morphology

Normally, the

sperm count

contains fewer

than 20%abnormal forms

e.g. bitailed,

short tailed, 2heads....etc.

Examples of abnormal sperm


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Examples of abnormal spermmorphology


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Other tests in semen analysis

Viability

When the motility is reported as less than 5% to10%, viability testing is recommended becauseprofoundly low motility may indicate dead sperm(necrospermia) .

The most common viability assessment involvesstaining with Eosin Y followed by counter

staining with Nigrosin. The viable sperm with itsintact cell membrane will not take up the dye andwill remain unstained.


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Hypo-osmotic swelling test (HOST)

an alternative method to assess sperm viability.

It is based on the principle that viable sperm

have intact cell membranes.

Exposure of the sperm to hypo-osmotic fluid will

cause water to flow into the viable cells seen asswelling of the cytoplasmic space and curling of

the sperm tail.

Nonviable sperm with nonfunctional cell

membranes will not exhibit this effect becausethey cannot maintain an osmotic gradient


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BIOCHEMICAL TESTS

Fructose in semen: Secreted from the seminal vesicle (150-650

mg%).

It is secreted for nutrition of sperm cells.

It disappears in cases of:

1. absence of seminal vesicle;

2. obstruction of ejaculatory duct;

3. inflammation of seminal vesicle. It is decreased in case of testosterone

deficiency.

fructose is used as fertility test. The used test is

Seliwanoffs.


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Acid phosphatase:

Secreted from the prostate.

The test is used as:

1. A marker ofprostatic functions2. In forensic laboratories as a test for the

presence of semen.


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Antisperm Antibody Testing:

Approximately 10% of infertile men will presentwith antisperm antibodies (ASA). Hence it has

been suggested to be tested routinely in all men

undergoing infertility work-ups.

Acrosin:

Low acrosin activity has been associated with

low sperm density, motility, and poor normal

morphology.


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Zinc:

It is necessary for chromatin stability anddecondensation, as well as forheadtaildetachment during fertilization.

L- carnitine:

It is secreted by the epididymis and isconcentrated in the seminal plasma at up to 10times the serum levels. It has a role in spermmaturation. Low L- carnitine levels are found in

oligoasthenozoospermic men.


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Alpha glucosidase:

This has been used to distinguish nonobstructive from obstructive azoospermia. It is

used as a specific marker for epididymal function

and is believed to play a role in sperm

maturation in the epididymis.A cutoff value of12 mIU/mL distinguishes ductal

obstruction from primary testicular failure.


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Normal semen parameters

Test Normal valuesLiquefaction Within 20 minutes

Morphology >70%normal,mature spermatozoa

Motility >60%

pH >7.0 (average 7.7)

Sperm count >20 million sperm/ml

Volume 1.5-5.0 ml

White blood cell < 1 million cell/ ml


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Precautions and steps of semensample collection

There should be 2 to 7 days of sexual

abstinence before collection. The duration

of abstinence should be constant, if

possible..

Two separate samples at least 7 days

apart should be analyzed.

It is best to collect the specimen in a clean(not necessarily sterile), wide-mouthed jar.


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It is important that the entire specimen be

collected, because the initial fraction

contains the greatest density of sperm.

Ideally, collection should take place in the

location where the analysis will be

performed.


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The degree ofsperm motility should bedetermined as soon as possible afterliquefaction, which usually occurs 15 to 20minutes after ejaculation

Semen should not be exposed to markedchanges in temperature, and if collected athome during cold weather, the specimen

should be kept warm during transport tothe laboratory.


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In order to allow liquefaction and mixing,semen is placed in a 370 C gently shaking

incubator for 30 minutes.

The semen sample should be examinedwithin 1 hour of production and receipt in

the laboratory.


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Clinical cases

Case 1 :

A 51 year old male with a history of 3 children in a priormarriage, an unremarkable medical history, and several(four) semen analyses that have revealed considerable

variability in terms of sperm concentration (12 million permL, 26 million per mL, 31 million per mL, and 94 millionper mL). The semen collections were all thought to becomplete and the other variables assessed in the semenanalysis (including motility and morphology) were

entirely normal. Question: What should be considered given this

information?


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Answer:

There is a normal variability in sperm

concentration for a normal fertile man. The

sperm concentration occasionally is decreasedeven in the normal fertile male population.

Therefore, the fact that most of the semen

analyses report a normal concentration is

encouraging. Also, the fact that this gentlemanhas proven fertility in the past is encouraging.


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Case 2:

A 38 year old male with a history of 2

children, an unremarkable medical history,

and a semen analysis that has revealed

persistent pyospermia (an excess number

of WBCs in the semen)

Question: What should be consideredgiven this information?


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Answer

Course of (broad spectrum) antibiotic

treatment should be given.


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Semen Practical Biochem