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 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

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

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

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

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 Menkveld etal., 1986; Menkveld, 2009

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 - 1987 3rd - 1992 4th - 1999 5th - 2010 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)

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) 16

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? 19

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 22

Acrosome reacted – Spermac stain 23

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

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

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

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

Cytoplasmic residues ROS production Immaturity of spermatozoa 28

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

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 ) 2000 2000 2000 2007 2007 Rhemrev et al., Unpublished data

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

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

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