1. Re-evaluation of sperm morphology assessment and results in light of new WHO (2010) manual reference limits
Roelof Menkveld, PhD
Andrology Laboratory, Department of Obstetrics and Gynaecology, Tygerberg Academic Hospital and University of Stellenbosch, Tygerberg, South Africa.
Pre-congress course: Cotemporary approaches in embryology laboratory – How can IVF success be raised?
III rd Congress of the Society of Reproductive Medicine
Cornelia Diamond Resort Belek, Antalya, Turkey
05 to 09 October 2011

2. Historical background of sperm morphology evaluation methodology
Basically two different evaluation approaches or methodologies
Liberal (old WHO) approach
Strict (Tygerberg criteria) approach

3. Early Liberal Approach
Normality for human spermatozoa
Based on approach in domestic animals with a homogeneous sperm population
Using modal forms of fertile males animals
In humans – heterogeneous picture
Thus not a feasible approach
Described abnormal spermatozoa based on consensus decision – thus normal by elimination
Morphological forms depicted by schematic and inaccurate drawings

4. Liberal approach Theoretical disadvantages
No specific criteria for normal
If not abnormal = Normal
Normal population will consist of
Abnormal population
True normal population
Therefore, can expect poor correlation with
Normal sperm function
Fertilisation and pregnancy rates

5. Liberal approach Disadvantages according to literature
Abnormal sperm morphology:
Is less sensitive for evaluation of ejaculate
Van Duijn et al., 1972
Has no correlation with pregnancy
Page and Holding, 1951
Of less importance compared to normal morphology
Hellinga, 1976

6. Strict Approach Conceptualized Biological based concept for normality
Late 1970’s early 1980’s
Tygerberg Hospital (R Menkveld)
Biological based concept for normality
Sperm selective capability of good cervical mucus 6

7. Strict (Tygerberg) criteria (1)
Whole spermatozoon must be considered
Head
Oval with smooth contours
Good distinction between acrosome and post acrosome region
Homogeneous light blue staining of acrosome 7

8. Strict (Tygerberg) criteria (2)
Correct neck implantation
No neck/midpiece abnormalities
No tail abnormalities
No cytoplasmic residues (>30% normal head)
NB - Borderline normal is abnormal 8

9. Literature on origin of Strict Criteria
Menkveld (1987)
The influence of environmental factors on spermatogenesis and semen parameters. PhD Dissertation. Faculty of Medicine, University of Stellenbosch, Tygerberg (Cape Town), South Africa.
Menkveld et al. (1990)
The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 5(5):

10. Evolution of sperm morphology evaluation approaches in consecutive WHO manuals
1980
Basic liberal approach
Very basic descriptions for normal spermatozoon
1987
Same basic approach
Slightly more descriptive information
1992
Strict approach should be followed
Borderline normal = Abnormal
1999 and 2010 (WHO-5)
Accept strict criteria - functionality based

11. Consequences of introduction of strict criteria

12. Overview of declining sperm morphology values over years
Menkveld etal., 1986; Menkveld, 2009

13. Cooper, 2007 (ESHRE campus meeting)
Normal values from WHO manuals, editions 2- 4 and lower reference limits from new 5th WHO manual (2010)
Semen parameter
WHO edition and year
2nd
3rd
4th
5th
Volume (ml)
2.0
1.5
Sperm concentration (106/ml) 20 15
Total sperm count (106) 40 39
Motility (% progressive) 50 28
Vitality (% live) 75 59
Morphology (% normal) 30
(15) 4
Cooper, 2007 (ESHRE campus meeting)

14. Possible reasons for lower normal sperm morphology values
Decline may be due to
Stricter application of (strict) sperm morphology evaluation criteria
Negative environmental influences
Recognition of additional sperm morphology abnormalities/parameters

15. Possible solution for declining normal sperm morphology values
In WHO-5 abnormal morphology group (≤ 3%)
Identification of
Additional abnormal sperm morphology patterns

16. Assessment of specific sperm morphology abnormalities
Four basic sperm abnormalities
Head abnormalities (Several classes)
Neck and midpiece abnormalities
Tail abnormalities
Presence of cytoplasmic residues
 Teratozoospermia index (TZI – WHO-5) 16

17. Head abnormalities can be used to determine abnormal sperm morphology patterns
Head abnormalities (Several classes)
Large
Small
Elongated (Tapering and pyriforms)
Acrosome abnormalities (Several classes)

18. Acrosome morphology classes
Differential classification of acrosomes
Normal
Staining defects
Too large
Too small
Other/Amorphous Total number of sperms with normal acrosomes
 Sperm morphology patterns

19. Are these specific abnormalities of any clinical significance?19

20. Large acrosomes – Spermac stain
Spontaneous acrosome reaction
No zona pellucida binding of spermatozoa

21. Small acrosomes
Mostly non-viable
Can not undergo acrosome reaction
Can not bind to zona pellucida

22. Acrosome reacted – Papanicolaou staining
Not able to bind to the zona pellucida 22

23. Acrosome reacted – Spermac stain23

24. Acrosomes with staining defects Beginning of acrosome reaction ?
Cysts and vacuoles ?
Membrane damage ?
Not able to bind to zona pellucida ?
DNA status (MSOME) ? 24

25. Large headed spermatozoa DNA status ?
Poor prognosis for normal in vitro fertilisation 25

26. Elongated spermatozoa pattern
DNA damage
Ultrastructural nuclear defects
Stress
Chromosome aneuploidy (Prisant et al., 2007) 26

27. Absence of centriole – no spindle formation in oocyte
Neck defects
Absence of centriole – no spindle formation in oocyte
Midpiece abnormalities
Mitochondrial defects (? Poor motility) 27

28. Cytoplasmic residues
ROS production
Immaturity of spermatozoa 28

29. Sperm morphology and fertilisation
Important aspects
Need morphological normal spermatozoa for normal sperm functions throughout the whole fertilisation pathway
Patients with clear abnormal sperm patterns
? Need for sperm functional tests
Patients with apparent high % normal morphology
? Sperm functional tests 29

30. Strict criteria still applicable?

31. Normal morphology distributions in 2000 vs 2007 (76 and 112 couples, mean normal morphology 7.3% and 7.2%; P= )
2000
2000
2000
2007
2007
Rhemrev et al., Unpublished data

32. Normal morphology: 2007 comparison between infertile and fertile population (n = 40 and 112)

33. Strict criteria still applicable?
Yes - with world wide co-operation
Problem
Lack off standardisation between different international QC schemes
Better Quality Control
Inter- and Intra-laboratory
Need international cooperation for
standardisation
Quality control

34. Thank you for your attention
Tygerberg Academic Hospital and University of Stellenbosch Medical school
Thank you for your attention 34