Sperm Factors on Embryo Implantation Potential

1. Sperm Factors on Embryo Implantation Potential Engin Enginsu M.D. Ph.D. Kadıköy Şifa Hospital, ART Unit Spermed Laboratory 2008

2. Semen Analysis • Minimum 2 sperm analysis • Ejaculate Volume 1.5 to 5.0 mL • Sperm Count >20 million/mL • Total Motility >30% • Progressive Motility >30% • Normal Morphology (MEUSC) >4% • Additionally • Agglutination None • Piospermia None • Viscosity Normal

3. Makler Counting Chamber

4. Semen Analysis • Phase contrast attachment • 20 x 10 magnification

5. Morphology • Head • Smooth oval shape • Acrosome 40-70% • Equatorial segment • Mid-piece • Tail • 10x100 magnification

6. Results of Semen Analysis n=2610 SPERMEDLaboratory 2008

7. Azoospermia • Centrifuge • Suspend pellet with 0,3 ml culture medium and vortex • All pellet should be evaluated on slide

8. Decrease in Sperm-ZP binding Oligozoospermia, severe teratozoospermia ZP-induced acrosome reaction Oligozoospermia ZP penetration problems Teratozoospermia Effects of Sperm Anomalies (Liu et al. 2004)

9. Acrosome Reaction • Acrosome is a membrane-bound cap-like structure covering the anterior part of the sperm head, containing: • Acrosine • Neuroaminidase • Hyaluronidase • Antibodies or Fluorescent probe • Vitality Test

10. Globozoospermia

11. Globozoospermia • Syndrome characterized by spherical chromatin structure and absence of acrosome • DNA structure unknown • Increase in sperm aneuploidy rate (case report) • Abnormal chromatin structure, DNA strand breaks • Diagnosed by morphological evaluation (Calogero et al. 1999 – 2001-2002)

12. Globozoospermia (Dam et al. 2007)

13. Globozoospermia • Responsible genes and inheritance is unknown • Heterologous ICSI: Centrosomal function defect, decrease in oocyte activation • Decrease in fertilization and oocyte activation • Pregnancy with ICSI, decreased fertilization rate • Calcium ionophore enhances fertilization rate (Dam et al. 2007, Rubes et al. 1998, Nakamura et al. 2002)

14. Globozoospermia • No spontaneous pregnancy • Aneuploidy rate increases in sex chromosomes and 13, 14, 15, 16, 18, 21 (Dam et al. 2007, Ditzel et al. 2005)

15. Macrosephalic Sperm

16. Macrosephalic Sperm and PGD (Kahraman et al. 2004)

17. Macrosephalic Sperm (Kahraman et al. 2004)

18. DNA Damage • DNA integrity is crucial for fertilization • Occurs in testicular, epididymal and post-ejaculatory levels • DNA strand breaks can cause chromosomal packaging abnormalities • High DNA damage ratio • Embryonic development arrests • Apoptosis • Implantation abnormalities • Early pregnancy loss (Evenson et al., 1999; Morris et al., 2002)

19. DNA Damage • DNA strand breaks are related with • Age • Schizophrenia • Achondroplasia • Apert's syndrome • Sperm count • Sperm motility • Blastocyst culture can be a good alternative to detect paternal inheritance in ART

20. DNA Damage (Lewis et al., 2005)

21. DNA Damage • Comet Assay(Single Cell Gel Electrophoresis Assay) • Sperm cell electrophoresis on agarose gel • Evaluation of DNA migration • Acridine Orange • Same principals with SCSA – Evaluation of results are difficult • Halosperm • Detection of DNA breaks - FISH • Halo evaluation after denaturation with acid and removal of nuclear proteins

22. ABNORMAL NORMAL Comet Assay

23. COMPt Green Fluorescence at Red Fluorescence DNA Damage • Sperm Chromatin Structure Assay (SCSA) • Acridine Orange Test, Aniline Blue • Fluorescent microscopy • Flow cytometry • Green normal, red DNA breaks • DNA Breaks = Red/Red + Green • More specific than Comet Assay and TUNEL Test • Advanced technology required (Lewis et al. 2008)

24. Halosperm (Sperm Dispertion Test) • Sperm heads with different sizes • DNA intact sperms sorted by size • Fragmanted DNA shows no halo in nuclear region Fragmanted Normal Enciso et al, 2006

25. TUNEL Test • Detection of single and double strand breaks in DNA • TdT-mediated dUTP nick end labeling (TUNEL) • Most common method for DNA break evaluation • Fast and easy Lewis et al, 2008

26. TUNEL: Terminal deoxynucleotidyl transferase mediated dUTP-biotin Nick End Labelling APOPTOTIC CHROMATIN NORMAL CHROMATIN

27. TESE: Alternative Approach • TESE + ICSI in high DNA breaks • Invasive • TUNEL: Reliability? • Sensivity and specificity??? • Cut-off value??? • Centrosome? (Greco et al., 2005)

28. Sperm Centriole Defects • Sperm centriole enters oocyte with fertilization, duplicates and forms sperm aster • Centriole abnormalities • Embryonic developmental defects • Implantation failures • Preclinical abortion (Van Blerkom, 1996 Rawe et al., 2002

29. Sperm Centriole Defects • Dithiothreitol (DTT) and Taxol restores sperm centriole function in Dysplasia of the fibrous sheath (DFS) • Reliability? (Heterologue ICSI/bovine-human) (Nakamura et al., 2005)

30. Sperm Centriole Defects • Post fertilization embryo development disorder or block • Sperm centriole in the sperm tail attachment site can not be transferred to active zygote centriole (Rawe et al., 2008)

31. Aneuploidy in Sperm • Sperm Chromosome Anomaly Ratio in patients with Normal Somatic Karyotype FERTILE ˜ %0,3 - 1,08 OLIGOZOOSPERMIA ˜ %0,7 - 9,44 TERATOZOOSPERMIA ˜ %1,3 - 3,90

32. Aneuploidy in Sperm • Although ICSI is a solution for male infertility, it causes inheritance of genetic abnormalities Non-obstructive Azoospermia 11.4-24.9 % Severe OAT 18 % Obstructive Azoospermia 1.8-5.8 % Ejaculatory Sperm 1.5-2.3 %

33. Aneuploidy in Sperm • Anomaly ratio in chromosomes X, Y and 18; • Sperm with Fragmented DNA v.s. Normal Sperm • 4.6 fold anomaly • Diploidy and disomy is 4.4 and 5.9 fold, respectively • Anomaly ratio in chromosomes X, Y and 17 • 1.5-4 times more in immature than mature sperms (Muriel et al., 2007)

34. Aneuploidy in Sperm • The frequency of aneuploidies, especially disomies, is significantly lower in sperm from the 80% Percoll fraction, that is, enriched in mature spermatozoa compared to the unprocessed semen sample. • Immature sperms have more fragmented DNA (Muriel et al., 2007)

35. Normal morphology X Y 24XX Sperm Sex Chromosome Disorders 18 • Infertile / Oligozoospermia • Increase in the number of sex chromosomes in sperm • Non-obstructive Azoospermia • Sex chromosome disorders in ~40 % of germ cells • Inheritance risk in most of the anomalies

36. Testicular Sperm • Sperm maturation • Sperm motility • Frozen sperm

37. Testicular Sperm • Fresh and frozen testicular sperm Fresh Frozen • Fertilization%79.3 %71.1 N.S. • Pregnancy %39.6 %25 N.S. • Implantation %25.4 %8.0 <0.05 • Live Birth %19.5 %6.0 <0.05 • De Croo et.al. 1998

38. Testicular Sperm • Motility in testicular samples • Motility r-FSH stim.Control • Fertilization %68.8 %42.1 • Implantation%20.1 %13.2 • Clinical pregnancy %47.9 %30 • Balaban et.al. 1999

39. Testicular Sperm • Increase in DNA damage in sperms, which are incubated or frozen, in Obsturictive Azoospermia patients • Incubation of testicular sperm is only beneficial for motility and morphology (Lewis et al., 2005, Dalzell et al. 2004)

40. DNA Damage in Testicular Sperm

41. High Magnification ICSI Hazout a. 2006, RBM Online

42. High Magnification ICSI Hazout a. 2006, RBM Online

43. Conclusions • Semen analysis reveals important information on genital and testicular function. • Fertilization disorders can be detected with sperm function tests. • Sperm DNA damage is related with reproductive disorders, however, insufficient for explaining all pregnancy losses.

44. Conclusions • Aneuploidy screening can be an alternative in abnormal semen analysis results. • Time dependant DNA damage can occur in testicular sperm. Thus, timing of ICSI after TESE should be shortened. • ICSI is not sufficient for sperm function disorders.

45. Conclusions • More studies needed for sperm centriole defect treatments. • Blastocyst culture is the most current treatment for paternal disorder eradication