Transcript No Slide Title

Semen analysis
What’s new
GAB 26/10/04
Ahmed Mahmoud, Frank Comhaire
Center for Medical & Urological
Andrology
UZ Gent
www.infertiliteit.net
www.andrology.be
1999
2000
Male reproductive tract
Ejaculatory sequence
Cowper’s glands & glands of ± 0.2 ml
Littré
Prostate, ampulla,
epididymis (+ spermatozoa)
± 0.5 ml
Seminal vesicles
2-5 ml
Aims of semen analysis

To discriminate between fertile semen and semen with
impaired fertilizing potential

To detect causal factors or mechanisms of impaired
fertilizing potential.

To direct treatment and predict treatment outcome
The semen sample
The semen sample









By masturbation (or Special Condom e.g. Male factor
Pack)
After 2-7 days of sexual abstinence
Within one hour of collection
If 1st abnormal then 2nd sample
Container:
clean, dry
warm (20-40°c) also during transport
Plastic: test for toxicity ‘Motility’
Wide-mouthed
Sperm counting
Reusable chambers
Disposable CC
Sperm concentration

Sample liquified, mix
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Liquefaction problem: use Bromelain (1g/l)
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Pipette: positive displacement
(at least for viscous samples)
bead
counted
concentration
(million/mL)
Counting chambers (beads)
55
50
45
Range ‡
Cost x 2
40
35
30
25
DROP
DROP1 Standard
count
Mahmoud et al. Fertil Steril 1997
Cell
vu
Cell
vision
Micro
cell
DEVICE
2X
cel
JCD
Makler
Bürker
Improved NEUBSPP†
Neubauer
Sperm concentration
Counting chambers (sperm)
(million/ml)
65
60
+SD § 55
50
Mean §
Ok
Best
45
40
-SD §
35
30
Experience +++
Experience ±
25
Cell
Vision
Mahmoud et al. Fertil Steril 1997
Makler
Bürker
Improved NEUSMP†
Neubauer
DEVICE
CONC‡
Sperm Motility
Sperm motility

Manual


Difficulties in quality control especially for grades A,
B
Accepted CV 10 % maximum

Automated systems:

Fully automated: Expensive, not problem-free

Semi-automated (Autosperm)
The AUTOSPERM
Start
A B C D E
1
2
3
4
5
Stop
Press
Button (4)
Sperm morphology
(Anton van Leeuwenhoek)
Liberal vs Strict criteria
Mahmoud A., Comhaire F.
Antwerp 15/10/02
Liberal criteria
Abnormal forms
Defined
Other
Spermatozoa
Normal
Phase contrast x 1000+
Grade a motility 42 %
Spontaneous pregnancy
Comhaire et al. (IJA 1987)
Morphology %
*
*
Spontaneous pregnancy
Menkveld et al. (HR 2001)
*
*
Determinants in-vivo
C: Comhaire 1988, M: Menkveld 2001
Variable
Sens.
Spec.
Grade a %
Grade (1-6)
Morphology
WHO
C 84
M 80
C 75
M 75
M 75
81
68
72
77
77
Morphology
strict
* Same criterion as IVF!!!
Criterion
value
42 %
4.5
42 %
30 %
4 %*
Grade 6>100µm/sec!!
Strict criteria & IUI pregnancy rate per cycle
Pregancy per cycle (%)
30
p=NS
n=91
25
n=267
20
n=53
15
10
5
0
0-4%A
Check et al., Arch Androl, 2002
5-14%
>14%
Morphology SC (% normal)
Morphology & IVF
Kruger et al. 1988
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Female (tubal) factor infertility
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Normal concentration >20 mil/ml, motility >30%
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Sperm morphology <14 % normal “strict criteria”
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Morphology of successful vs. failed IVF
a. Normal
b. +/“slightly amorphous”
c. Abnormal
“severely amorphous”
Kruger
et al.
1988
Morphology & IVF
Kruger et al. 1988
50
Successful
40
Failed
R2=0.56
R2=0.36
30
20
10
R2=0.44
0
Normal
Strict&WHO
“Slightly amorphous”
Strict=Abnormal
WHO=normal
Mophology
index
=WHO
Morphology & IVF
Host et al. 1999 (Acta Obstet Gynecol Scand)

100 couples (50 tubal factor, 50 unexplained)

Technician blinded

WHO criteria better predictor for fertilization
rate than Kruger's criteria (p<0.002)
Morphology CV%: 1 sperm
misclassified
Method & Reading 1 Reading 2 CV %
number
% normal % normal
Strict 100
14
15
4.88
Strict 200
14
14.5
2.5
WHO 100
30
31
2.3
CV of different methods of sperm morphology
More Liberal
Strict
50
45
40
CV %
35
30
25
20
15
American
Society
Clinical
Pathology
50 %
33 %
* CV is even
higher
Without
transformation
21 %
10
5
0
ASCP
Keel et al., HR 2000
WHO
Method
Strict*
* After data transformation
I quote Rune Eliasson, Androlog mail, 16.09.02
• “Classification of sperm morphology according to
'strict criteria' has been accepted by gynecologists and
many others to a degree that is totally unfounded.
• It has become a new paradigm and will take a
considerable amount of work and time to get rid
of”.
N. B. Kruger (1986) morphology is a modification of
the methods by Eliasson !! (1971) & MacLeod (1962)
Conclusions (morphology)

Use high magnification, good optics (phase
contrast x1000+)
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Define your own normal values
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Morphology less important in vivo

Be liberal, Use liberal criteria
Immunological infertility
The role of antisperm antibodies in male
infertility
Spermatozoa “loaded” with
spermagglutinins stick to the
glycoprotein filaments as soon as
they come with contact with
cervical mucus.
Cervical mucus containing
spermagglutinins provides the
penetrating spermatozoa with the
spermagglutinins and afterwards the
spermatozoa stick to the glycoprotein
filaments.
Diagnosing Immunological
Infertility
Antibodies in semen: IgG and IgA-class
 Antibodies in serum: agglutinating,
cytotoxic (requiring complement)
 Current techniques in routine analysis of
semen and serum

Detecting Antisperm Antibodies
attached to Spermatozoa
(direct tests)
Mixed Antiglobulin Reaction (MAR)
Schematic representation of the direct
MAR test
Immunobead test
Detecting Antisperm Antibodies in
Serum
(indirect tests)
Indirect SpermMAR test
Antisperm Antibodies
Immunobead
Motility
rapidly
SpermMAR
good
Preparation
time
non
S. Volume
0.5-2.0 ml
10 µ l
Shelf life
1 month
1 year
Sensitivityspecificity
Price
Better
~X2
2. Reactive oxygen species
& WBCs
Secretory Products of the accessory
Sex Glands
Refining the Diagnosis of MAGI
Prostate
Citric acid
Acid phosphatase
Gamma-glutamyl-transferase
Zinc-calcium
pH (acidic)
Liquefaction
Epididymis
Seminal vesicles
Semen volume
pH (alkaline)
Alpha-glucosidase
Coagulum
L-carnitine
Fructose phosphoryl choline
Glyceryl
Prostaglandins
Antioxidants
Assessing the Function of the
Epididymides
Alpha-glucosidase and sperm
fertilizing potential
Milingos et al. (1996) Eur.J.Obstet.Gynecol.Reprod.Biol
64, 115
In IUI more pregnancies occurred when
markers of epidydimal function (Shorr stain
& alpha-glucosidase) were normal (OR:
11.1, CI: 2.1-59).
Criterion value: 78 mIU/ejaculate
Alpha glucosidase and sperm concentration
Alpha glucosidase (mU/L)
120
100
80
60
40
20
ND
AZOOSPERMIA
(n=67)
OLIGOZOOSPERMIA NORMOZOOSPERMIA
(n=216)
(n=189)
Difference between groups: p<0.05 (ANOVA)
ALPHA-GLUCOSIDASE (mU/L)
Alpha glucosidase in azoospermia
30
crit <=13.5 U/L
sens: 82 %
spec: 70 %
25
20
15
10
N
D
Epididymal
(caput)
(n=5)
Functional Vasectomy
azoospermia
(n=33)
(n=27)
Epididymis and Antioxidants

Antioxidants are produced in the same region of
the epididymides that produce alpha-glucosidase

Semen with low alpha-glucosidase activity
presents high oxidative overload
Alpha-glucosidase: correlations
n
r
p
Gamma-GT
384
0.62
<0.001
WBCs (mill/ml)
Cases > 1 mill/ml
ROS
165
-0.30
<0.001
104
-0.27
<0.001
Assessing the Function of the Prostate
and/or the Seminal Vesicles
Localizing the affected organ(s) in
MAGI
Markers of accessory sex glands in semen
Ejac. volume
Fructose (conc.)
GT/Citric acid
-glucosidase
Prostatitis
Prostato
vesiculitis
=
=
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=
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
=
Prostato Epididymitis
epididymitis
=
=
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=
=
=
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Effect on treatment: Choice of antibiotic, Use of antioxidants