final seminal fluid analysis
TRANSCRIPT
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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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%icroscopic e*amination
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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).
%icroscopic e*amination
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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.