re-evaluation of sperm morphology assessment and results in light of new who (2010) manual reference...

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

Historical background of sperm morphology evaluation methodology

Basically two different evaluation approaches or methodologies – Liberal (old WHO) approach– Strict (Tygerberg criteria) approach

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

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

Liberal approachDisadvantages 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

• Page and Holding, 1951• Hellinga, 1976

Strict Approach

• Conceptualized – Late 1970’s early 1980’s

– Tygerberg Hospital (R Menkveld)

• Biological based concept for normality– Sperm selective capability of good cervical

mucus

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

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

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):586-92.

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

Consequences of introduction of strict criteria

Overview of declining sperm morphology values over years

70

60

50

40

30

20

10

0

Year of semen analyses

Morp

holo

gy (

% n

orm

al)

1968

1970

1972

1974

1976

1978

1980

1982

1984

1986

1988

1990

1992

1994

1996

1998

2000

2002

2004

2006

2008

Menkveld etal., 1986; Menkveld, 2009

Normal values from WHO manuals, editions 2- 4 and lower reference limits from new 5th WHO manual (2010)

Semen parameterWHO edition and year

2nd - 1987 3rd - 1992 4th - 1999 5th - 2010

Volume (ml) 2.0 2.0 2.0 1.5

Sperm concentration (106/ml) 20 20 20 15

Total sperm count (106) 40 40 40 39

Motility (% progressive) 50 50 50 28

Vitality (% live) 50 75 75 59

Morphology (% normal) 50 30 (15) 4

Cooper, 2007 (ESHRE campus meeting)

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

Possible solution for declining normal sperm morphology values

In WHO-5 abnormal morphology group (≤ 3%)

Identification of

Additional abnormal sperm morphology patterns

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)

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)

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

Are these specific abnormalities of any clinical significance?

Large acrosomes – Spermac stain

• Spontaneous acrosome reaction

• No zona pellucida binding of spermatozoa

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

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

Acrosome reacted – Spermac stain

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

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

Elongated spermatozoa pattern

• DNA damage

• Ultrastructural nuclear defects

• Stress

• Chromosome aneuploidy (Prisant et al., 2007)

Neck defects• Absence of centriole – no spindle formation in oocyte

Midpiece abnormalities• Mitochondrial defects (? Poor motility)

Cytoplasmic residues

• ROS production

• Immaturity of spermatozoa

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

Strict criteria still applicable?

Normal morphology distributions in 2000 vs 2007 (76 and 112 couples, mean normal morphology 7.3% and 7.2%; P=0.5443 )

25

20

15

10

5

0

MORPH NormF

Rhemrev et al., Unpublished data

2000 200720002000 2007

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

Norm

25

20

15

10

5

0

Diagnosis

0 1

>3.0Sens: 87.5Spec: 85.0

Infertile Fertile

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

Tygerberg Academic Hospital and University of Stellenbosch Medical school

Thank you for your attention