1. Semen analysis What’s new GAB 26/10/04
Ahmed Mahmoud, Frank Comhaire
Center for Medical & Urological Andrology
UZ Gent

2. 1999
2000

3. Male reproductive tract

5. Ejaculatory sequence Seminal vesicles 2-5 ml
Cowper’s glands & glands of Littré
± 0.2 ml
Prostate, ampulla, epididymis (+ spermatozoa)
± 0.5 ml
Seminal vesicles
2-5 ml

7. 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

8. The semen sample

9. 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

11. Sperm counting

12. Reusable chambers

13. Disposable CC

15. Sperm concentration Sample liquified, mix
Liquefaction problem: use Bromelain (1g/l)
Pipette: positive displacement
(at least for viscous samples)

16. Mahmoud et al. Fertil Steril 1997
counted bead
concentration
(million/mL)
Counting chambers (beads) 55 50 45 40 35 30 25
DROP DROP1 Standard Cell Cell Micro X JCD Makler Bürker Improved NEUBSPP†
count vu vision cell cel Neubauer
Range ‡
Cost x 2
Mahmoud et al. Fertil Steril 1997
DEVICE

17. Mahmoud et al. Fertil Steril 1997
Sperm concentration
(million/ml)
Counting chambers (sperm) 65 60
+SD § 55 Ok 50
Best 45
Mean § 40 35
-SD § 30
Experience ±
Experience +++ 25
Cell
Vision
Makler
Bürker
Improved
Neubauer
NEUSMP†
CONC‡
Mahmoud et al. Fertil Steril 1997
DEVICE

21. Sperm Motility

23. Sperm motility Manual Automated systems:
Difficulties in quality control especially for grades A, B
Accepted CV 10 % maximum
Automated systems:
Fully automated: Expensive, not problem-free
Semi-automated (Autosperm)

24. The AUTOSPERM

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

28. Sperm morphology (Anton van Leeuwenhoek)

29. Liberal vs Strict criteria
Mahmoud A., Comhaire F.
Antwerp 15/10/02

30. Liberal criteria
Abnormal forms
Defined
Other
Spermatozoa
Normal
Phase contrast x 1000+

32. Spontaneous pregnancy
Grade a motility 42 %
Spontaneous pregnancy
Comhaire et al. (IJA 1987)
Morphology %

33. Spontaneous pregnancy* *
Spontaneous pregnancy
Menkveld et al. (HR 2001) * *

34. Determinants in-vivo * Same criterion as IVF!!! Grade 6>100µm/sec!!
C: Comhaire 1988, M: Menkveld 2001
Variable
Sens Spec.
Criterion
value
Grade a %
Grade (1-6) C M
42 %
4.5
Morphology WHO C M
30 %
Morphology
strict
4 %*
* Same criterion as IVF!!! Grade 6>100µm/sec!!

35. Strict criteria & IUI pregnancy rate per cycle30 25 20 15 10 5
p=NS
n=91
n=267
n=53
Pregancy per cycle (%)
0-4%A
5-14%
>14%
Morphology SC (% normal)
Check et al., Arch Androl, 2002

36. Morphology & IVF Kruger et al. 1988
Female (tubal) factor infertility
Normal concentration >20 mil/ml, motility >30%
Sperm morphology <14 % normal “strict criteria”
Morphology of successful vs. failed IVF

37. a. Normal b. +/- “slightly amorphous” c. Abnormal Kruger et al. 1988
“severely amorphous”
Kruger
et al.
1988

38. Morphology & IVF Kruger et al. 198850
Successful
R2=0.56
Failed 40
R2=0.36 30
Morphology % normal 20
R2=0.44 10
Normal
Strict&WHO
“Slightly amorphous”
Strict=Abnormal
WHO=normal
Mophology
index
=WHO

39. 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)

40. Morphology CV%: 1 sperm misclassified
Method &
number
Reading 1
% normal
Reading 2 % normal
CV %
Strict 100 14 15
4.88
Strict 200
14.5
2.5
WHO 100 30 31
2.3

41. CV of different methods of sperm morphology
More Liberal
Strict 50
50 %
* CV is even higher
Without transformation 45
American
Society
Clinical
Pathology 40 35
33 % 30
CV % 25 20
21 % 15 10 5
ASCP
WHO
Strict*
* After data transformation
Method
Keel et al., HR 2000

43. 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)

44. Conclusions (morphology)
Use high magnification, good optics (phase contrast x1000+)
Define your own normal values
Morphology less important in vivo
Be liberal, Use liberal criteria

46. Immunological infertility
The role of antisperm antibodies in male infertility

47. 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.

48. 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

49. Detecting Antisperm Antibodies attached to Spermatozoa (direct tests)

50. Mixed Antiglobulin Reaction (MAR)

51. Schematic representation of the direct MAR test

53. Immunobead test

55. Detecting Antisperm Antibodies in Serum (indirect tests)

56. Indirect SpermMAR test

57. Antisperm Antibodies Immunobead SpermMAR Motility rapidly good
Preparation
time
non
S. Volume ml
10 µ l
Shelf life
1 month
1 year
Sensitivity-specificity
Better
Price
~ X 2

58. 2. Reactive oxygen species & WBCs

61. Secretory Products of the accessory Sex Glands
Refining the Diagnosis of MAGI

62. Prostate Epididymis Seminal vesicles Citric acid Acid phosphatase
Gamma-glutamyl-transferase
Zinc-calcium
pH (acidic)
Liquefaction
Seminal vesicles
Semen volume
pH (alkaline)
Coagulum
Fructose
Prostaglandins
Epididymis
Alpha-glucosidase
L-carnitine
Glyceryl phosphoryl choline
Antioxidants

63. Assessing the Function of the Epididymides

64. 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: ).
Criterion value: 78 mIU/ejaculate

66. Alpha glucosidase and sperm concentration
120
100 80
Alpha glucosidase (mU/L) 60 40 20 ND
AZOOSPERMIA
(n=67)
OLIGOZOOSPERMIA
(n=216)
NORMOZOOSPERMIA
(n=189)
Difference between groups: p<0.05 (ANOVA)

67. ALPHA-GLUCOSIDASE (mU/L)
Alpha glucosidase in azoospermia 30
crit <=13.5 U/L
sens: 82 %
spec: 70 % 25 20
ALPHA-GLUCOSIDASE (mU/L) 15 10 N D
Epididymal
(caput)
Functional
azoospermia
Vasectomy
(n=27)
(n=5)
(n=33)

68. 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

69. Alpha-glucosidase: correlations
Gamma-GT
384
0.62
<0.001
WBCs (mill/ml)
Cases > 1 mill/ml
165
-0.30
ROS
104
-0.27

70. Assessing the Function of the Prostate and/or the Seminal Vesicles

72. Localizing the affected organ(s) in MAGI

73. Markers of accessory sex glands in semen
Prostatitis Prostato Prostato Epididymitis
vesiculitis epididymitis
Ejac. volume =  = =
Fructose (conc.) =  = =
GT/Citric acid    =
-glucosidase = =  
Effect on treatment: Choice of antibiotic, Use of antioxidants