1. How to diagnose and treat male infertility in 2011 Roelof Menkveld, PhD Andrology Laboratory, Department of Obstetrics and Gynaecology, Tygerberg Academic Hospital and University of Stellenbosch. III rd Congress of the Society of Reproductive Medicine Cornelia Diamond Resort Belek, Antalya, Turkey 05 to 09 October 2011

2. Lecture objectives Give an overview of the evolution of the (normal) semen parameter values of the different WHO manual editions from 1980 to 2010 (WHO-5) Discuss the usefulness of the new semen parameter values with special reference to normal sperm morphology of WHO-5 Discuss some treatment options in cases where men present with poor semen analysis results

3. Old manuals Old wording in previous manuals –1st edition No specific wording or definitions for semen parameter values ( Used normal and fertile range) –2nd and 3rd editions Used term “normal” values –4th edition uses term “reference” values Statement –The (mean?) normal “reference” values quoted in these manuals are for “normal” men and NOT the MINIMUM requirements for fertilisation

4. New wording for definition of “normal” values in 5th WHO manual edition New wording for “normal of reference” values –Refer to Lower reference limits Reference ranges

5. Material and methods for WHO-5 edition (1) Reference population –Fathers (Couples with time to pregnancies of ≤ 12 months) –1600+ couples –Five centres from 3 continents Samples –Only 1 sample per father –Complete sample after 3-7 days of abstinence WHO manual, 2010

6. Material and methods for WHO-5 edition (2) Methods –Only laboratories following WHO manual guidelines Sperm concentration by haemocytometer only –Sperm morphology evaluation according to STRICT CRITERIA only Statistics –Reference values based on the lower 5th percentile limits WHO manual, 2010

7. Normal values for WHO manuals, editions 2- 4 and expected lower reference limits and WHO- 5 manual Semen parameter WHO edition and year 2nd - 19873rd - 19924th - 19995th - 2010 Volume (ml)2.0 1.5 Sperm concentration (10 6 /ml)20 15 Total sperm count (10 6 )40 39 Motility (% progressive)50 28 Vitality (% live)5075 59 Morphology (% normal)5030(15) 4

8. Recent studies proposing new “cut-off, normal or reference” values Three types of literature studies –Based on In vivo or in vitro pregnancies Fertile versus subfertile populations Lower interval values

9. Lower percentile intervals Ombelet et al., 1997 - Lower 10th percentile Menkveld et al., 2001 - Lower 10th percentile Haugen et al., 2006 - Lower 10th and 5th percentile

10. Comparison of expected new WHO manual lower reference values and recent published values Semen parameter Publication Menkveld et al., 2001 * Haugen et al., 2006 5th WHO manual 5th10th Sperm concentration (10 6 /ml) N/A10.616.915 Motility (% progressive) 20334328 Morphology (% normal) 3344 * Adjusted ROC curve values

11. Comments on the lower reference values of WHO-5 manual Has lead to more confusion especially with clinicians Need a more “precise or detailed” breakdown of semen parameter values Need a new approach to interpretation of normal sperm morphology values

12. Need for a more “precise or detailed” breakdown of semen parameter values Use of categories or intervals

13. Classification of male fertility potential according to semen parameters as used at Tygerberg Hospital Semen parameter Fertility potential classification InfertileSubfertileFertile Concentration (10 6 /ml)< 2.02.0 – 9.9≥ 10.0 Motility (% progressive)< 1010 – 29≥ 30 Morphology (% normal)< 55 - 14≥ 15 Semen volume (ml)< 1.0> 6.01.0 – 6.0 Fertile or Normal = Optimal chance for pregnancy Subfertile or Borderline = Reduced chance for pregnancy Infertile or Pathological = Small change for pregnancy Menkveld, 2007

14. Need for a new approach for the interpretation of normal sperm morphology values

15. Sperm morphology Values as determined by strict (Tygerberg) criteria (Menkveld et al., 1990) and according to the old editions of WHO manuals are not applicable anymore due to decrease in normal sperm morphology values over years New approach for interpretation of sperm morphology parameters is needed Menkveld et al., 1990

16. Overview of declining sperm morphology values over years Menkveld etal., 1986; Menkveld, 2010

17. Prognostic sperm morphology groups Currently used based on strict criteria (Menkveld et al., 1990) Normal –≥ 15% morphological normal spermatozoa Good prognosis group –5 to 14% morphological normal spermatozoa Poor prognosis group –≤ 4% morphological normal spermatozoa WHO-5 lower (Normal) reference value –≥ 4% morphological normal spermatozoa Menkveld et al., 1990; Kruger et al., 1986; Kruger et al., 1988; WHO, 2010

18. Declining normal sperm morphology values Decline due to three possible reasons –Stricter application of evaluation criteria –Negative environmental influences –Additional parameters for sperm morphology abnormalities

19. Stricter application of sperm morphology evaluation criteria Introduction of STRICT CRITERIA –Strict versus liberal approach Chanced from borderline spermatozoa previous regarded as normal to TOO BE REGARDED AS ABNORMAL –Over critical approach for interpretation of normal –Inadequate training

20. Negative environmental influences Exposure to pseudo-estrogens of mother, unborn baby and male –Higher incidences of decrease in male reproductive health Higher exposure to toxic environment and occupation hasards –Decrease in spermatogenesis and lower/poorer semen parameters Higher incidences of sexual transmitted diseases –Lower semen parameters –Increase of leukocytospermia –Increased sperm DNA damage

21. Decline due to introduction of additional parameters for sperm morphology abnormalities For example –Differential classification of acrosome morphology Normal Staining defects Too large Too small Other/Amorphous

22. New approach for interpretation of sperm morphology parameters is needed Better use of existing sperm morphology parameters Better quality control Use of additional sperm morphology parameter, especially in patients with teratozoospermia according new lower reference value of ≤ 3% (Poor prognosis group)

23. Better use of existing sperm morphology parameters Acrosome morphology (Acrosome index) –TZI –Cytoplasmic residues –Semen cytology Identification, reporting and treatment of WBC on semen smears

24. Better quality control for sperm morphology evaluation Problem Lack of intra- and inter-laboratory quality control Lack off standardisation between different international QC schemes Solutions Betters adherence to WHO guidelines (aim of new WHO manual) Better co-operation between and standardisation of the different international QC schemes

25. Use of additional sperm morphology parameters In WHO abnormal morphology group (≤ 3%) Identification of abnormal sperm morphology patterns –Abnormal acrosome staining –Large sperm/acrosome patterns –Small sperm/acrosome patterns –Elongated sperm morphology patterns

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

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

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

29. Acrosome reacted – Spermac stain

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

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

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

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

34. Cytoplasmic residues ROS production Immaturity of spermatozoa

35. Theoretical treatment option ( Male fertile) Semen parameter Fertility potential classification InfertileSubfertileFertile Concentration (10 6 /ml)< 2.02.0 – 9.9≥ 10.0 Motility (% progressive)< 1010 – 29≥ 30 Morphology (% normal)?? ≤ 3 ≥ 4 Semen volume (ml)< 1.0> 6.01.0 – 6.0 Treatment will depend on several factors Age of couple – ART for older women Years of infertility – If woman is “normal” give time If female factor treat with ART

36. Theoretical treatment option ( Male subfertile) Semen parameter Fertility potential classification InfertileSubfertileFertile Concentration (10 6 /ml)< 2.02.0 – 9.9≥ 10.0 Motility (% progressive)< 1010 – 29≥ 30 Morphology (% normal)≤ 3???? ≤ 3#≥ 4 Semen volume (ml)< 1.0> 6.01.0 – 6.0 ?? ≤ 3# - No apparent abnormal sperm morphology pattern Treatment will depend on several factors Age of couple – ART for older women (IUI, IVF) Years of infertility – If woman is “normal” give limited time If female factor - treat with ART (IVF, ICSI)

37. Theoretical treatment option ( Male infertile) Semen parameter Fertility potential classification InfertileSubfertileFertile Concentration (10 6 /ml)< 2.02.0 – 9.9≥ 10.0 Motility (% progressive)< 1010 – 29≥ 30 Morphology (% normal)?? ≤ 3 ≥ 4 Semen volume (ml)< 1.0> 6.01.0 – 6.0 ?? ≤ 3# - IF apparent abnormal sperm morphology pattern Treatment = ICSI ??

38. Tygerberg Academic Hospital and University of Stellenbosch Medical School, Tygerberg (Cape Town), South Africa Thank you for your attention

39. References Haugen et al., Int J Androl 27:66-71,2006 Kruger et al., Fertil Steril 46:1118-23,1986 Kruger et al., Fertil Steril 49:112-7,1988 Menkveld R. In: Male infertility – Diagnosis and treatment. Oehninger and Kruger (eds). Informa Healthcare, 2007 Menkveld et al., Human Reprod 5(5):586-92,1990 Menkveld R. Asian J Androl 12(1):47-58,2010 Menkveld et al., Arch Androl 17:143-4,1986 Menkveld et al., Hum Reprod 16:1165-71,2001 Ombelet et al., Hum Reprod 12:987-93,1997 World Health Organisation. WHO laboratory manual for the examination and processing of human semen. WHO Press, Geneva. 2010