3/2/2009

Semen analysis
Wh ’ new
What’s 1999 2000

Male reproductive tract

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Ejaculatory sequence

Cowper’s glands & glands of ± 0.2 ml

Littré
Prostate ampulla,
Prostate, ampulla ± 0.5 ml
epididymis (+ spermatozoa)

Seminal vesicles 2-5 ml

Aims of semen analysis

„ To discriminate between fertile semen and semen with

impaired fertilizing potential
The semen sample
„ To detect causal factors or mechanisms of impaired
fertilizing potential.

„ To direct treatment and predict treatment outcome

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The semen sample

ƒ By masturbation (or Special Condom e.g. Male factor

Pack)
ƒ After 2-7 days of sexual abstinence
ƒ Within one hour of collection
ƒ If 1st abnormal then 2nd sample
ƒ Container:
„ clean, dry
„ warm ((20
20--40°
40°c) also during transport
„ Plastic: test for toxicity ‘Motility’
„ Wide--mouthed
Wide

Reusable chambers

Sperm counting

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Disposable CC

counted bead
concentration Counting chambers (beads)
Sperm concentration (million/mL)
55

50

„ Sample liquified, mix 45

Cost x 2
40
ge ‡

„ Liquefaction
Li f ti problem:
bl use Bromelain
B l i (1
(1g/l)
/l)
Rang

35

30

„ Pipette: positive displacement 25

(at least for viscous samples) DROP DROP1 Standard
count
Cell
vu
Cell
vision
Micro
cell
2X
cel
JCD Makler Bürker Improved NEUBSPP†
Neubauer
Mahmoud et al. Fertil Steril 1997 DEVICE

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Sperm concentration
(million/ml) Counting chambers (sperm)
65

60

+SD § 55
50
Ok
45
Best
Mean §
40

35
-SD §
30
Experience ± Experience +++
25
Cell Makler Bürker Improved NEUSMP† CONC‡
Vision Neubauer
Mahmoud et al. Fertil Steril 1997 DEVICE

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Sperm Motility

The AUTOSPERM
Sperm motility
„ Manual

¾ Difficulties in quality control especially for grades A,

B
¾ Accepted CV 10 % maximum
„ Automated systems:

¾ Fully automated: Expensive, not problem-

problem-free

¾ Semi--automated (Autosperm)
Semi

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Start

A B C D E
1
2
3
4
5
Stop
Press
Button ((4
4)

Sperm morphology
(Anton van Leeuwenhoek)
Leeuwenhoek)

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Liberal criteria

Abnormal forms

Defined
Liberal vs Strict criteria
Other

Spermatozoa

Normal
Phase contrast x 1000+
1000+

Grade a motility 42 %

Spontaneous pregnancy
Comhaire et al. (IJA 1987)
1987)

Morphology %

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*
* Determinants in
in--vivo
Spontaneous pregnancy C: Comhaire 1988,
1988, M: Menkveld 2001
Menkveld et al. (HR 2001)
2001) Variable Sens. Spec. Criterion
value
Grade a % C 84 81 42 %
*
Grade ((11-6) M 80 68 4 .5
Morphology C 75 72 42 %
WHO M 75 77 30 %
* Morphology M 75 77 4 %*
strict
* Same criterion as IVF!!! Grade 6>100
100µm/sec!!
µm/sec!!

Strict criteria & IUI pregnancy rate per cycle Morphology & IVF
Kruger et al. 1988
30
n=91 p=NS
„ Female (tubal) factor infertility
per cycle (%)

25
n=267
20
n=53 „ Normal concentration >20
>20 mil/ml, motility >30
>30
30%%
15
Pregancy p

10

5 „ Sperm morphology <

<14
14 % normal “strict criteria”
0
0-4%A 5-14% >14%
„ Morphology of successful vs. failed IVF
Morphology SC (% normal)
Check et al., Arch Androl, 2002

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Morphology & IVF

Kruger et al. 1988
a. Normal 50
Successful
R2=0.56
40 Failed
R2=0.36
b. +/-
+/-
30
“slightly amorphous
amorphous”
20

c. Abnormal 10
R2=0.44
“severely amorphous”
amorphous”
0
Normal “Slightly amorphous” Mophology

Kruger et al. 1988 Strict&WHO Strict=Abnormal

WHO=normal
index
=WHO

Morphology & IVF Morphology CV%: 1 sperm

Host et al. 1999 (Acta Obstet Gynecol Scand)
misclassified
„ 100 couples (50
(50 tubal factor, 50 unexplained)
Method & Reading 1 Reading 2 CV %
number % normal % normal
„ Technician blinded
Strict 100 14 15 4.88

„ WHO criteria better predictor for fertilization Strict 200 14 14.

14.5 2.5
rate than Kruger's criteria (p<0
(p<0.002
002))
WHO 100 30 31 2.3

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CV of different methods of sperm morphology

More Liberal Strict
50
45
50 % * CV is even
American
40
Society
higher
35
Clinical Without
30
Pathology
33 % transformation
f i
CV %

25
20
15
21 %
10
5
0
ASCP WHO Strict*
Keel et al., HR 2000 Method * After data transformation

I quote Rune Eliasson

Eliasson,, Androlog mail, 16.
16.09
09..02
Conclusions (morphology)
• “Classification of sperm morphology according to „ Use high magnification, good optics (phase
'strict criteria' has been accepted by gynecologists and contrast xx1000
1000+)
+)
many others to a degree that is totally unfounded.
„ Define your own normal values
• It has become a new paradigm and will take a
„ Morphology less important in vivo
considerable amount of work and time to get rid
of””.
of
„ Be liberal, Use liberal criteria
N. B. Kruger ((1986
1986)) morphology is a modification of
the methods by Eliasson !! (1971
(1971)) & MacLeod (1962
(1962))

11

Spermatozoa “loaded” with
spermagglutinins stick to the
glycoprotein filaments as soon as
they come with contact with
cervical mucus.
3/2/2009
Immunological infertility
The role of antisperm antibodies in male
infertility
Diagnosing Immunological
Infertility
„ Antibodies in semen: IgG and IgA
IgA--class
„ Antibodies in serum: agglutinating,
gg g,
cytotoxic (requiring complement)
„ Current techniques in routine analysis of
Cervical mucus containing semen and serum
spermagglutinins provides the
penetrating spermatozoa with the
spermagglutinins and afterwards the
spermatozoa stick to the glycoprotein
filaments.
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Mixed Antiglobulin Reaction (MAR)

Detecting Antisperm Antibodies

attached to Spermatozoa

(direct tests)

Schematic representation of the direct

MAR test

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Immunobead test

Indirect SpermMAR test

Detecting Antisperm Antibodies in
Serum

(indirect tests)

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Antisperm Antibodies
Immunobead SpermMAR
Motility rapidly good
Preparation time non
2. Reactive oxygen species
S. Volume 0.5-2.0 ml 10 µ l & WBCs
Shelf life 1 month 1 year
Sensitivity-
Sensitivity- Better
specificity
Price ~X2

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Prostate

Citric acid
Acid phosphatase
Gamma-glutamyl-transferase
Zinc-calcium
Secretory Products of the accessory pH (acidic)
Liquefaction
Sex Glands
Refining the Diagnosis of MAGI Epididymis
Seminal vesicles
Semen volume
pH (alkaline)
Alpha-glucosidase
Coagulum
L-carnitine
Fructose phosphoryl choline
Glyceryl
Prostaglandins
Antioxidants

Alpha-glucosidase and sperm

Alpha-
fertilizing potential
Milingos et al. ((1996
1996)) Eur.J.Obstet.Gynecol.Reprod.Biol
Assessing the Function of the 64,, 115
64
p y
Epididymides In IUI more ppregnancies
g occurred when
markers of epidydimal function (Shorr stain
& alpha-
alpha-glucosidase) were normal (OR:
11..1, CI: 2.1-59).
11 59).
Criterion value: 78 mIU/ejaculate

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Alpha glucosidase and sperm concentration

120

ucosidase (mU/L)
100

80

60

Alpha glu
40

20

ND

AZOOSPERMIA OLIGOZOOSPERMIA NORMOZOOSPERMIA

(n=67) (n=216) (n=189)

Difference between groups: p<0.05 (ANOVA)

Alpha glucosidase in azoospermia

COSIDASE (mU/L)

30
crit <=
<=13
13..5 U/L Epididymis and Antioxidants
25 sens: 82 %
spec: 70 %
20
„ Antioxidants are produced in the same region of
15
the epididymides that produce alpha-
alpha-glucosidase
ALPHA-GLUC

10
N
D
„ Semen with low alpha-
alpha-glucosidase activity
presents high oxidative overload
Epididymal Functional Vasectomy
(caput) azoospermia
(n=27
(n=27))
(n=55)
(n= (n=33
(n=33))

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Alpha--glucosidase: correlations
Alpha

n r p
Assessing the Function of the Prostate
Gamma--GT
Gamma 384 0.62 <0.001
and/or the Seminal Vesicles
WBCs (mill/ml) 165 -0.30 <0.001
Cases > 1 mill/ml
ROS 104 -0.27 <0.001

Localizing the affected organ(s) in

MAGI

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Markers of accessory sex glands in semen

Prostatitis Prostato Prostato Epididymitis

vesiculitis epididymitis

Ejac. volume = Ì = =
Fructose (conc.) = Ì = =
γGT/Citric acid Ì Ì Ì =
α-glucosidase = = Ì Ì

Effect on treatment: Choice of antibiotic, Use of antioxidants

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