What do we mean by
semen collection errors??
Have you had
any occasion when the concerned husband failed to procure fresh semen when
trigger has been done and there is failure to comply with collection of seems
at the time of IUI?? “Situational anejaculation”??
Every media and is ready but there is
collection problems in IUI? Magnitude of
the problem?? How big is the problem??
-
10
% of men are unable to give a semen sample
-
Some have never
masturbated
-
Some are inhibited in the clinic(unfamiliar situation) .
-
Some fail
on demand (DEMAND EJACULATION) .
-
Some
are brought up at religious family and cant masturbate.
-
Quite
often semen spills in collection room error and
few man are too anxious and mixes tap water to make the total volume
identical to normal ejaculate vol of 2-5 ml.
-
Few
men use saliva/ some lubricating agent to ease the masturbation and this can
falsely decrease the count.
-
-
Failure to
procure a sample can be very
embarrassing and disheartening for a
man and can produce long delays in treatment/ cancellation of the
cycle unless frozen sample is ready well ahead which is costly . To avoid this problem always ask whether a man can collect a sample when
advising a semen test. If he reports difficulty in collecting semen
instruct him to try collecting at home
using coitus interrupts . Special non spermicidal condoms are also available. Those who are still unable to collect a sample will
usually be able to ejaculate when stimulated with a vibrator .
Message on male subfertility :--1) It is important
to remember that semen analysis is only a rough predictor of fertility 2) So reassurance is very important when the semen is sub normal because
the male ego is very threatened
by a poor semen report 3) It is not
uncommon to see that many pathology laboratories still quote the old
norms misleading the doctor and making the couple
unnecessarily nervous.4)
Many men with poor semen
parameters will still father
children while others with
seemingly normal parameters will be
infertile. 5) The duration of
infertility should be considered in addition
to the semen analyses. If the duration
is short then even with a low count
the chances of pregnancy are
good while if the duration of infertility is long then
even with a normal analysis the chances of pregnancy are poor.
Time acts as a filter sorting
out the fertile from the sub fertile regardless of the semen
parameters Hence young
couples who have been trying only for a short time should not rush into advanced
reproductive techniques.
Azoospermia
is present in 1% of all men and 10-15%
infertile men. To establish diagnosis of azoospermia :
Semen specimen is centrifuged
at high speed and pellet is examined at magnification of 400 X.
Note : Absence of
sperms should be documented on
two occasions to diagnose
azoospermia .
Azoospermia
can be
Obstructive : Blockage anywhere in the ductal system from efferent ductules to ejaculatory ducts as a consequence of :
Severe infection
Iatrogenic injury
during scrotal or inguinal surgery
Congenital absence
of vas.
Intrauterine
Insemination
Non obstructive : It may be due to :
· Intrinsic testicular disease
· Endocrinopathies or other conditions that suppress spermatogenesis
Note : Men with non
obstructive azoospermia should have a careful examination of centrifuged sample as in one third of these men
a very low sperm production
insufficient to drive epididymal
transport and entry to ejaculate is
present . These sperms can be
recovered by TESE for ICSI. If the case the case under discussion is a confirmed case
of OLIGO,& urosurgron has examined and excluded anatomical diseases
-endocrine evaluation, including PRL, Cortisol, DHEAS-O4,TSH, Renal, Metabolic
profiles and karyotyping has been done and H/O drug intake and life style have
been duly analyzed then there are several options depending on the age of
female partner, duration of marriage etc. If female partner is >35 yrs and
duration-of marriage is > 10-14 yrs, I shall prefer for ICSI
straightway.-because from social point of view upbringing of child usually
takes about long 15 yrs active life on the mothers side. But this ICSI is
applicable if count is persistently below< 10 million. I understand many
will opt for IUI with oral antioxidants,/ pooling of several samples-->IUI,
but as because the female partner is aged I personally is biased for
ICSI/traditional IVF-ET. The other option particularly for relatively young
couple, say wife is somewhere between 30-32 yrs-then possibly the oral
antioxidants , modified life style of husband , avoiding illicit drugs may be
attempted. In such settings IUI has a definite place provided the count is 5-15
million with over 30% motility and at least 608 % morphology normal. But when husband comes to know that
his count is low he, willfully refrain from coitus in the IUI cycle so that a
good no. of sperms will be available for IUI procedure. This should be strongly
discouraged. Result of IUI will be low.. I must admit, I have no personal
experience of treating male partner --with HCG/hMG .not to speak of CC.My
mentor, Prof. B N Chakrovorty used to prescribe Methyl Testosterone Tab during the period 1065-1085 when I was
associated with him and worked under his supervision. But , If count is
below< 5 million ,I routine perform Karyotyping and exclude Fragile X
syndrome/ CF gene mutation if couple can afford. before I proceed for ART
procedures(be it ICSI/IVF-ET). I have no experience in prescribing on
Tamoxifene, /Inj testosterone- but now I am impressed with results of
antioxidants particularly those containing Vit E, C B12 selenium.
What
are the
·
Physical properties of semen ?? A) opacity:-- If semen looks absolutely transparent (like
water) then there is reason to believe that count is really too low. But after
due liquefaction if it (liq sample) is reasonably turbid then it implies a
fairly good amount of sperms must be present to make the liq. sample(
crude sample) turbid in naked eye/ or at least opaque. .Otherwise it would have
been as clear as water provided there is absence of pus cells in light
microscopy. This observation may give a clue about oligozoospermia or
azoospermia
·
·
B) Viscosity: A highly viscous sample may falsely cause OLIGOZOSPERMIA (low density) semen-A) if semen
is viscous- a primary disease of prostate/ seminal vesicle-has to be considered
due to lack of hyaluronidase enzyme. However in a viscous ample too actual count will be apparently low if the technician
is not dedicated & do not know how to assess the count in a viscous semen
. . Prostate secrets PAP, Vesiculase,
Hyaluronidase enzymes etc to liquefy the coagulum formed at upper vagina soon
after ejaculation which coagulum sticks to the upper vagina loosely.,Threby
even if female partner leaves bed for toilet or dress change most of the ejaculated semen remain stuck at
upper vagina entrapped in coagulum.. The
semen coagulum forming after natural ejaculation -on the ectoCx/ upper vagina-
after deposition in the female genital tract starts liquefying after about
10-20 minutes and then though most of the sperms at the periphery of coagumlum
are dead or devoid of motility die to acid pH. We should remember that sperm
cells are the most fragile cells of body and Oocytes and anterior horn cells of
spinal cord arte the largest cells of the body and none of last two cant
regenerate once damaged..
Low density if associated with
low volume semen implies collection errors, frequent coitus/
diseases of accessory glands ( to be confirmed by clinical exam/ TRUS(transrectal
Ultra sonography) / Clinical evaluation by Urologist).
What else to order
in oligozoospermia?? Presence
of blood/urine & Bilirubin may be ordered if persistently report come as
oligozoospermia .Incomplete ejaculation
occurring due ,out of apprehension in uncommon settings--- may be rare cause of
oligozoospermia so also unilat ejaculatutory duct obstruction
(semen of pH will be fall below 7). .
How useful is clinical
examination?? Therefore it is mandatory to
clinically examine the male partner before embarking on costly tests-
karyotyping, endocrine evaluation, TRUS etc/ Colour Doppler not to speak of
antioxidant/ Antibiotic Ry.
Relevance of PCT in
2020?? I personally still insist on
POST COITAL TESTS in low density sperm count cases( 7 motile sperms/HPF) is
excellent and defies the Lab report of oligozoospermia.. Sometimes things
become easy .
Pus cells may not be pus
cells : No unnecessary antibiotics please:-Incidentally
pus cells and immature sperms are not usually differentiated with appropriate
stain (peroxidase stain but not by H E stain) neither agglutination of sperms
are duly tested. Regards-Dr. S K Pal.
For kind attention of junior
members only:-There several books available on male infertility formulating tr.
plans for male subfertility .. I quote herewith which may be of help to U. A)
Male subfertility . -A clinical guide-Ed, Anne M Jwequier, Cambridge University
Press(www.cambidge.org) B) Male Infertility-
Contemporary Clinical Approaches. Andrology, ART& Antioxidants-SPRINGER,
Ed. Sijo J. Parekttil , Ashok Agarwal. C) Infertility in the Male- 4th Ed. Ed
Larry I Lipshultz, Cambridge University Press(www.cambidge.org);
D) Male reproductive Dysfunction Author- S C Basu, Jaypee) Additionally the
Biennial Review of Infertility Vol 3, 2013, (Springer Publisher) -the very
first chapter is dedicated on role of antioxidants in male subfertility..Dr
Pal.
SPERM MOTILITY
Sperm motility is assessed as a percentage of total
sperm population exhibiting any motion. Factors which
effect sperm motility are :
Patients age,
health status, and length of
time last ejaculated
Patients
exposure to outside influences
such as excessive
heat or toxins
Length of time and
adequacy of handling from collection to
analysis.
Forward progression
Whereas sperm motility represents the
quantitative parameter of
sperm movement expressed
as a percentage sperm progression represents the quality
of sperm movement expressed in a subjective scale.
Forward progression is graded on a scale of 0 to 4
0: No motion
1: Motion with no forward progression
2 : Erratic movement
with slow forward progression
3: moderate speed with
relatively straight forward motion
4: Rapid forward
progression
The motility of each sperm is further graded a to d
Poor sperm motility
may be due to :
Testicular dysfunction
Anti sperm
antibody
Genital tract infection
Partial obstruction of
ejaculatory duct
Varicocele
Prolonged abstinence
Immotile cilia syndrome
Intrauterine Insemination
Total
progressive motility
Percentage of sperms exhibiting
purposeful forward motion . The probability
of conception increases with increasing motility upto 60%
Motility assessment
can be done in two ways
1.A wet
preparation
2.Computer aided sperm
analysis.
Six uL of undiluted well shaken semen
is put on a clean slide and
covered with a cover slip. This gives
a standard depth of observation.
Examination should begin after one minute
when flow has stopped.
Motile sperms are defined as any sperm with a moving flagellum whether or not the sperm is
progressive.
Number of fields to be examined: At least 5 randomly selected fields are sampled
Number of sperms to be counted : 200 spermatozoa are counted for motility and classified.
The counting procedure is repeated
in the second chamber.
Difference in counts between two
chambers : if the percentage of difference
between the two values is :
10% or lesser the mean
of the two values is taken
More than 10% a third reading is taken and an average of the three is taken.
CASA
The CASA instrument digitalizes the electrical signals that result from repeated video scans of a field of sperm The sperm cell is recognized
because of its optical
contrast with the background.
CASA provides numbers
that quantitative the vigor and
pattern of movement on a per sperm
basis.
When no motile sperms are observed a sperm viability test should be done to differentiate viable non motile sperms from dead sperms
as it is important to identify living
non motile sperms for ICSI
Sperm morphology
Normal sperm morphology has been related to fertilizing
potential of sperm. This may be because
:
1.Abnormal sperms inability to deliver
genetic material to cytoplasm of the egg.
2.Sperms are more likely
to have diminished motility due to :
3.Hydrodynamic inefficiency of head shape
4.Abnormalities in tail structure which prevent normal motion
5.Deficiency in energy production which
is necessary for movement
Do not bind to the
zona as well as normal sperms.
Sperm morphology
usually is assessed from seminal
smears that are prepared at the time of semen evaluation and subsequently stained . It can be assessed in several ways the most common classification system being
the third edition WHO standard
and the Kruger strict criterion .
BASIS: Spermatozoa with visible anatomical defects are not functional cells and there
are many clinical reports that associate defective sperm shapes
with infertility . There is
considerable variation in
visual morphology assessment . According
to the WHO k standards the cut off for normal
semen forms is 30% . This was
decreased to 14%
Whereas sperm
concentration and progressive
motility have value in discriminating fertile and infertile
men strict sperm morphology has
emerged as the one most
discriminating value.
Strict sperm morphology
represents the best predictor
of sperm function is a widely
accepted indication for fertilization
by ICSI.
The likelihood of male infertility was increased approximately 4 fold when strict normal
sperm morphology was less than 9% . The 9%
threshold value has a sensitivity of 43%
and specificity of 815 for identifying infertile men. When sperm
morphology is assessed according
to strict normal standard in vitro fertilization efficiency
correlated with percentage
of normal sperms
during IVF.
Method of preparation
The WHO method
requires either a wet slide
preparation or a fixed stained slide. A
10 to 20 ul drop of semen
is prepared on a slide. After
placing a cover slip over
the specimen morphology may be assessed
Alternatively the specimen may be fixed
with equal volume of fixative and Methylene blue prior to fixing it on the slide. At least two smears
can be made from fresh semen sample.
Effect of method
of preparation on results. The
method of preparation has an effect on results. Sperm dimensions
are different on seminal
smears in comparison with wet smear and staining
methods can affect cell classification . The greatest accuracy and precision on morphometric measurement are obtained when sperms are washed
and resuspended to standard
concentration before slide
preparation
If sperm concentration is > 20 x10 ml 5 ul semen is used
If concentration is <
20 x10 ml 10- 20 ul semen is used.
They are fixed and stored for staining
Normal morphology
For a sperm to be considered normal the sperm
head neck midpiece and tails should be normal and single.
Method : Examination is done in oil immersion at 100x field
magnification . An ocular
micrometer is essential for
thorough morphological examination .
Defects are expressed per 100
sperms for that region.
Number of sperms to be
assessed : 200 sperms are assessed . Only recognizable
spermatozoa with tails are considered in the count. Immature cells upto the stage of round spermatids are not
counted . 100 consecutive sperms are counted
in two randomly selected areas
If percentage difference between the two is :
· Less
than 15% then the mean of the two is recorded as percentage morphology
· More than 15% a third reading is taken and average
of all three is taken.
Protocol for
evaluation :
Criterion for further evaluation is a single
head and tail. Sperms with more
than one head or tail are scored as
duplicate sperms and are not evaluated
further . so also those without head
classified as headless or pinhead
sperms . Sperms with any tail
abnormality are classified as
amorphous tail regardless of head
or midpiece morphology . The rationale for primacy of tail
morphology is that such sperms can be considered dysfunctional because absent or severely abnormal flagellar activity will result
in failure of sperm transport
to site of fertilization or penetration of oocyte. If sperm tails
are normal in size and shape
the head of the sperm is considered. Midpiece morphology is assessed
last only if head and tail are normal . To be classified as normal the sperm must be normal in all three – head tail and
midpiece.
There may be
sperms with more than one abnormality
Teratospermic index-Average number of sperm defects per sperm
1.WHO
criterion for sperm morphology
WHO criterion for assessing sperm morphology includes the following :
Head
Defects
· Oval , smooth head normal
Round
Pyriform , pin , double , and amorphous head is abnormal Head Defects
The length of the head should be 4.0-5.0 um and width 2.5-3.5 um.
The length to width ration should be 1.50 to 1.75
There should be a well defined pale acorsomal region comprising 40-70 % of the head
area
The sperm head appears pale blue
in the Acrosomal region and dark blue
around
·
Tail Defects
· Single , unbroken straight without kinks or coils
· A
normal semen analysis
should contain 30%
normal sperms using
WHO criterion
· Krugers strict criterion
· In order to perform
the Kruger strict criterion
sperm morphology is evaluated by placing 5 ul of liquefied semen on a slide making a thin smear and air drying at room temperature . The slide is then fixed
and stained Kruger
criteria for assessing normal forms
include the following
Midpiece Defects
Midpiece should be slender
less than 1 um in width and one
and a half times in length of head and axially attached to it . Midpiece
· Straight slightly thicker
than the tail
Cytoplasmic droplets should be less than half the size of the normal
head and are stained green Papanicolaou
stain is used.
The midpiece is stained red.
Tail
The tail should be uniform straight thinner than midpiece uncoiled
and approximately 45 um long attached symmetrically
The tail is either blue or red
This classification requires that
all sperms which are not clearly normal are
considered abnormal . The forms are considered normal If > 14% are normal .
Kruger strict criterion
for morphology
Classification
Normal - > 14%
Borderline – 4 to 14%
Abnormal - < 4 %
Abnormal :
The defect could be in the
head midpiece or tail
Tail defects : The defect seen are
:
Short
Multiple
Hairpin
Broken tails
Bent tails
Tails of irregular width
Coiled tails
Combination of these
A high number of coiled
tails indicate that the sperms have been subjects to hypo
osmotic stress. It may also
be due to sperm ageing . If >
20% it must be noted. Sperms with any tail abnormality
are classified as amorphous tail regardless of head or midpiece morphology
Head defects : These are :
Large
Small Tapered
Round
Amorphous heads
Vacuolated heads
Head with small acorsomal
area
Double heads
Combination of these
Pin head or micro head sperm are
not counted
Neck and midpiece defects
Bent neck
Asymmetrical insertion of
the midpiece into head
Thick and irregular midpiece
Abnormal thin midpiece
Combination of these
Cytoplasmic droplets : Cytoplasmic
droplets are more than one third
the area of normal head sperm. They are usually located
in the midpiece.
Method of Measurement
A transparent overlay is drawn using a stage micrometer to determine the size of the boxes. The
overlay is placed on the screen so that
the base of the sperm head is aligned with the bottom of the
overlay.
Normal : If the length and width lie between the boxes the
classification is normal
Large : If the length and
width lie outside the outer box the
classification is large.
Small : If the length lies within
the inner box it is small
Tapered : if the length lies
outside the outer box or between the two boxes and the width inside the inner box it is
tapered
Amorphous : If the outline of the
sperm head is irregular
or asymmetrical or both the
classification is amorphous regardless
of sperm dimension . If the outline is
smooth and symmetrical but the length lies between
the boxes and width outside
the outer box it is termed amorphous
Kruger strict method has been
used to predict a patient’s
fertility to show the most
consistent prediction of fertilization and in vitro
following insemination. This method of assessing normal sperm morphology because
of its precise non subjective nature establishes
a threshold value below
which abnormal morphology
becomes contributing factor to infertility .
Cellular elements other than
spermatozoa
The ejaculate contains cells other than spermatozoa referred to as round cells. These include
germinal line cells
sloughed from seminiferous tubules
epithelial cells from
genitourinary tract prostate cells
spermatogenic cells and leukocytes . A normal ejaculate should not contain more than 1x10 round cells
Printed :IUI Tips.
Good semen preparation should have 6-8 million/ml of
prepared semen and 99% motility.
Double IUI only when perifollicular PSV (Peak Systolic
Velocity):- > 20-25 cm/sec.
Good success of IUI depends on good USGà Color Doppler/Power Doppler/ 3D power
Doppler of follicles and endometrial blood flow.
CO2 incubator must, :IUI in the second cycle of
post-drilling.
Prevention of OHSS.
If at the time of IUI the serum E2 is > 5000 pigmy/ml
then possibility of IUIà immediately
treat with IV albumin & start cabergolin 0.5 mg for 8 days.
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