final seminal fluid analysis

8/18/2019 Final Seminal Fluid Analysis

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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 one of the first tests done tohelp determine whether a man has a problem fathering a

child (infertility).

• A problem with the semen or sperm affects more than

one-third of the couples who are unable to have

children (infertile).


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• Sperm is the male gamete, that is, the male se cell, which hasthe capacity to fertilise an egg. Sperm are produced in

the seminiferous tubes of the testes. Germ cells for the

production of spermatozoa

• Speciali!ed Sertoli cells provide support and nutrients for thegerm cells as they undergo mitosis and meiosis

(spermatogenesis).

• "hen spermatogenesis is complete, the immature sperm

(nonmotile) enter the epididymis.

• #n the epididymis, the sperm mature and develop flagella.

• $hey remain stored in the epididymis until e%aculation.

• $he e%aculatory ducts receive both the sperm from the ductus

deferens and fluid from the seminal vesicles.

Physiology


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Fluid Fractions

1. Urethral glands (&-') are very small mucussecreting glands.

2. Prostate: Approimately & to * of the semen

volume is acidic fluid produced by the prostate gland,the secretion contains acid phosphatase and

proteolytic en!ymes that act on the fluid from theseminal vesicles, resulting in the coagulation and

liquefaction of the semen.


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3. Seminal vesicles (produce about +- of the fluid

volume of semen) viscous, yellowish secretion is rich

in fructose, vitamin , prostaglandin, and other

substances, which nourish and activate the sperm

passing through the tract.

4. estis ! "pididymis: (') Spermato!oa are produced

in the testis under the influence of testosterone, and

then the epididymis (is the first part of the duct

system) provides a temporary storage site for the

immature sperm that enter it from testis. $his fraction

still in the inactive form until e%aculation.


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Formation o# the sperm cell

• Spermatogenesis is a cascade of cell divisions/

o0itosis/ spermatogonia to primary spermatocytes

o 1irst meiotic division/ secondary spermatocytes

o

Second meiotic division/ haploid spermatids• $his process ta2es 3 4 + days in the human.

• Spermiogenesis: differentiation of the round spermatidinto a spermato!oon


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Sperm transport and seminal plasma

• 5$esticular sperm6 need to undergo more maturationsteps before they are ready to fertili!e.

• $ransported from the testes to the epididymis, where

they mature, and acquire the ability to swim.

• $hen moved to the vas deferens, for storage.

• At e%aculation, the sperm are transported out of the

vas and mi with accessory gland secretions/

7 prostatic fluid (p8 slightly al2aline to neutral9contains citric acid and !inc).

7 seminal vesicle fluid (p8 strongly al2aline9

contains fructose).


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$hat the spermatozoon

• $he human sperm cell is about 3 :mlong.

• $he nucleus is in the head 7 contains

the &* chromosomes.

• #t is the head which binds to the egg at

fertili!ation.

• 0id-piece/ the energy for motility is

generated.

• $ail/ (motility the beat is initiated %ust

behind the mid-piece, and then

propagated along the tail).


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$hat the purpose o# the test

• #nvestigation of fertility• #dentify treatment options

Surgical treatment.

0edical treatment.

Assisted conception treatment.

;etermine the suitability of semen for artificial insemination

or #


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Sample collection

• Specimen should be collected into pre warmed (&=o), sterile,

non-toic, wide-mouth container, after a couple has abstain fromseual activity for &-* days to not longer than ' days.

• Specimens collected following prolonged abstinence tend to

have higher volumes and decreased motility.

• "hen performing fertility testing, two or three samples areusually tested at &-wee2 intervals, with two abnormal samples

considered significant.

• $he specimen should be delivered to the laboratory within =

hour of collection and the laboratory personnel must record thetime of specimen collection and specimen receipt.

• $he sample must be 2ept at *3> until analysis, which begins

ideally within * min, but absolutely within min, of

e%aculation


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%ethods o# collection

1. Masturbation (the method of choice for all seminal fluidtests).

2. By condom/ it is not recommended for fertility testing

because the condoms may contain spermicidal agents.

3. By coitus interrupts/ (withdrawal method).4. TESA/ $esticular sperm etraction ($?S?)- @pen $esticular

iopsy/ is a highly invasive, open surgical procedure

performed under general anaesthetic. $he scrotum and testes

are cut open, before testicular tissues are cut away andeamined for sperm, which, if present can be etracted.


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&a'el o# sample

• Batient name• linic number

• ;ate and time

•Caboratory request form

• he #ollo(ing should 'e recorded on the la'oratoryanalysis #orm)

7 $he period of abstinence (in days). 7 #f sample collection was complete or incomplete.

7 $he time interval from collection to analysis


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%acroscopic e*amination

• $here are several macroscopic evaluations which give useful

diagnostic information about the sample/

Appearance

@dour

Ciquefaction


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&i+ue#action• A fresh semen specimen is clotted and should liquefy

within * to minutes after collection9 therefore,recording the time of collection is essential for

evaluation of semen liquefaction.

• Analysis of the specimen cannot begin until after

liquefaction has occurred.

• #f after & hours the specimen has not liquified

proteolytic en!ymes such as alpha-chymotrypsin may

be added to allow the rest of the analysis to be

performed.


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%acroscopic e*amination

,olume/ normal is (&-' mC). Dsing disposablevolumetric pipette .

• "8@ criteria specify that any volume greater than

&. mC is normal. Cow volume may indicate partial or

complete bloc2age of the seminal vesicles, or that theman was born without seminal vesicles.

• ,iscosity) ?stimate the viscosity of the semen byaspirating the semen into the measuring pipette and

allowing the semen to drop by gravity and will not

appear clumped. @bserve the length of the thread.

volumetric pipette


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• $he normal p8 of semen is al2aline with a range of 3.to ..

• #ncreased p8 is indicative of infection within the

reproductive tract.

• A decreased p8 is associated with increased prostatic

fluid.

pH


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%icroscopic e*amination

• he characteristics assessed are)

0otility.

Sperm aggregation (random clumping)/ 5some6 is normal, but

large clumps (each with hundreds of sperm) is abnormal.

Spermagglutination (between specific sites)/ could suggest the presence of antisperm antibodies.

?pithelial cells/ usually present in small numbers

?rythrocytes/ should not be present

acteria and proto!oa/ presence indicates infection


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-ormal Semen nalysis

Semen volume &ml or more (usually &-+ milliliters per e%aculation)

Semen p/ Semen p8 of 3.&-.

&i+ue#action time &-* minutes after collection

Sperm count + million spermato!oa per e%aculate or more

Sperm morphology 0ore than * of the sperm have normal shape andstructure.

Sperm motility 0ore than ' of the sperm show progressivemovement or &' or more with rapid progressivemovement.

,itality 3' or more live, i.e., ecluding dye

$hite 'lood cells 1ewer than = million "sEml


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• Formal values for sperm concentration are commonly listed as

greater than & million sperm per milliliter , with concentrations between = and & million per milliliter considered borderline.

• $he total sperm count for the e%aculate can be calculated by

multiplying the sperm concentration by the specimen volume.

• $otal sperm counts greater than + million per e%aculate are

considered normal (& million per milliliter & mC).



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%ethods o# measuring sperm concentration

• /emacytometer) Sperm can be counted by ma2edilution =/& in $0 pipette or by automatic pipette

(which is more accurate) with a solution containing

sodium bicarbonate ('g) and formalin (=ml)

(immobili!e G preserve the spermato!oa), tap water(= ml) will suffice as a diluent.


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Sperm concentration haemocytometer

• $he number of squares assesseddepends on the number of sperm

counted in the first large square/

7 #f H = counted, the whole

grid is assessed

7 #f =-+ counted, = squares

are assessed

7 #f I + counted, ' squaresare assessed

First large square counted

large central square.

$his square is ruled into &' small squares,

each of which is further divided into =


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Sperm concentration calculations

• #f the counts of the two chambers are not within ' of theiraverage discard, remi the sample, and set it up again

• #f the two counts are in agreement, then the sum of the two

counts is divided by the correction factor/

#f & J &' squares counted, divide their sum by =

#f & J = squares counter, divide their sum by +

#f & J ' squares counted, divide their sum by &

• $his gives the sperm concentration in millions per ml

• Sperm count K concentration J total volume


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alculations

• Dsing a =/& dilution and four large "Ls squarescounted

• $he sperm concentrationEml K Fo of sperms counted

',

• Dsing a =/& dilution and five small MLs squares

counted

• $he sperm concentration Eml K Fo of sperms

counted =,,


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Sperm concentration interpretation

• $he "8@ Meference values for/

Sperm concentration is I & J =, spermEml

• ounts of less than & million per milliliter (H&

millionEml) are considered sub-fertile• #f a man has a sperm concentration H ' J =, spermEml,

the "8@ recommends assessment for numerical and

structural abnormalities of se chromosomes


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irect smear or $et preparation

Blace =:l of thoroughly mied, liquefied semen on aclean glass slide under a lightly applied glass cover

slip will allow visuali!ation of the sperm in a

specimen of semen under 8B1.


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• $he "orld 8ealth @rgani!ation has a value of ' andthis must be measured within minutes of collection.

• A man can have a total number of sperm far over the

limit of & million sperm cells per milliliter, but stillhave bad quality because too few of them are motile.

• $he other way around, a man can have a sperm count

far less than & million sperm cells per millilitre andstill have good motility, if more than of thoseobserved sperm cells show good forward movement.

%otility


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%otility o# sperm are divided into #our di##erent grades

• Grade 4) Sperm with progressive motility. $hese are thestrongest and swim fast in a straight line. Sometimes it is alsodenoted motility a.

• Grade 3) (non-linear motility)/ $hese also move forward but

tend to travel in a curved or croo2ed motion. Sometimes alsodenoted motility b.

• Grade 2) $hese have non-progressive motility because they donot move forward despite the fact that they move their tails.

• Grade 1) $hese are immotile and fail to move at all .


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Sperm morphology

• 0orphology is even more important than motility and

concentration

• ecause of the small si!e of the human sperm head, must use anair-dried smear which has been stained

• Brepared samples are assessed using a =J oil-immersionob%ective under bright field optics

• $he "8@ recommends that & spermato!oa are counted persample

• 1ields for counting must be selected at random

• "hen counting, remember about the normal distribution


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'normal morphology


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Sperm ,itality

• Dsually performed using a vital stain, such as eosin N,with a counterstain (nigrosin) to differentiate live

(unstained) and dead (stained) spermato!oa.


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gglutination

• Agglutination of spermato!oa means that motile spermato!oastic2 to each other, head to head, mid-piece to mid-piece, tail to

tail, or mied, e.g. mid-piece to tail.

• $he adherence of either immotile or motile spermato!oa tomucus threads, to cells other than spermato!oa, or to debris is

not considered agglutination and should not be recorded as such.

• $he presence of agglutination is suggestive of an immunologicalfactor of infertility.


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5ther cells in semen

• &eu6ocytes) normally (=-+E8B1), increase number(leu2ocytospermia) indicates reproductive tract infection

• "pithelial cells) normally (=-&E8B1)

• Spermatocytes) (#mmature germ cells) =-&E8B1.

• "rythrocytes) (=-&E8B1). #ncreased number may indicate areproductive tract infection or damage to a small capillary duringsample production.

• -ote) bacteria and proto!oan such as $richomonas vaginalis areuncommon in human semen but their presence is indicative of possible male reproductive tract infection and should be reportedto the referring doctor for further evaluation.


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Semen 'iochemistry

• cid phosphatase) mar2er for prostatic function• itric acid) can indicate prostatic function 7 low

levels may indicate dysfunction or a prostatic duct

obstruction

• 7inc) mar2er for prostatic function 7 colorimetric

assay (WHO)

• Fructose) mar2er for seminal vesicle function,and is a substrate for sperm metabolism 7

spectrophotometric assay (WHO)


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erms

• spermia) absence of semen

• zoospermia) describe a total absence of spermato!oain semen. (After centrifuge sperm count is !eroE8B1).

• 5ligozoospermia) refers to a reduced number ofspermato!oa in semen and is usually used to describe a

sperm concentration of less than & millionEml. Spermcount '-= spermE8B1.

• Severe oligospermia) sperm count =-& spermE8B1.

• Polyzoospermia) denotes an increased number ofspermato!oa in semen and is usually refers to a spermconcentration in ecess of *' millionEml.


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• Asthenozoospermia: refers to a man who produces a

greater proportion of sperm which are immotile or

have reduced motility, compared to the "8@ referencevalues.

• Teratozoospermia: sperm carry more morphological

defects than usual


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7I

• $he eratozoospermic Inde* is an epression of the averagenumber of abnormalities per abnormal sperm

• ?ach sperm cell is assessed for an abnormality in the head,nec2Emid-piece, or tail, and for a cytoplasmic droplet

• #f it does not have any of these abnormalities, it is 5normal6• #f it does have an abnormality, it is 5abnormal6, and we score

each abnormality. So, if a cell has an abnormal head and tail,it is counted as = cell, and & abnormalities

• $hen, (total O abnormalities) E (total O sperm) K $P#

• A $P# I =. has been associated with poor sperm fertili!ingability


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in#ertility


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$hat is in#ertility8• #nfertility means not being able to get pregnant after one year of

trying. @r, women who can get pregnant but are unable to stay pregnant may also be infertile.

• re!nancy: is the result of a process that has many steps. $o get

pregnant/

A woman must release an egg from one of her ovaries(ovulation).

$he egg must go through 1allopian tube toward the uterus

(womb).

A manQs sperm must %oin with (fertili!e) the egg along the way.

$he fertili!ed egg must attach to the inside of the uterus

(implantation).

#nfertility can happen if there are problems with any of these

steps.


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• Hi!h temperature o" testic#es $testes%. Sperm are made in the

testes which are in the scrotum. $his is the bodyQs way of

2eeping the testes slightly cooler than the rest of the body,

which is best for ma2ing sperm.

• Smo&in! can a""ect the sperm count. #f you smo2e, you

should stop completely for optimum sperm production.

• A#coho#: equivalent eight pints of normal strength beer or

siteen small glasses of wine, may interfere with optimum

fertility.

• Medicines and dru!s. 0ost do not interfere with sperm

production but some may do. $hese include/

tetracyclines, , colchicine,allopurinol.

$hat increases a man9s ris6 o# in#ertility?


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$hat things increase a (oman9s ris6 o#

in#ertility8

Age

Stress

Boor diet

eing overweight or underweightSmo2ing

?cess alcohol use

Seually transmitted infections (S$#s)

8ealth problems that cause hormonal changes, such as

polycystic ovarian syndrome and primary ovarian

insufficiency


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assisted reproduction techni+ues

:;<


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• AM$ includes all fertility treatments in which both eggs and

sperm are handled. #n general, AM$ procedures involve surgically

removing eggs from a womanLs ovaries, combining them with

sperm in the laboratory, and returning them to the womanLs body.or procedures in which a woman ta2es medicine only to stimulate

egg production without the intention of having eggs retrieved.

• he di##erent types o# assisted reproductive technology

:;


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2. 7ygote intra#allopian trans#er $*')T%: is similar to #


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Sperm preparation

• ,iscontinuous density !radient: is a technique inwhich all sperm in the seminal fluid are separated by

centrifugation.

• S-im up: is a technique in which the most motile

sperm are selected from the e%aculate. 1resh semen iscovered with a medium and placed in a cylinder which

is laid at a +'o angle. 0otile sperm in the e%aculate

will then swim up to the top of the tube, leaving

immotile sperm and debris in the lower part of the

cylinder.

final seminal fluid analysis

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