Sperm Preparation in High DFI | Infertility Treatment | Jindal IVF Chandigarh

1. Sperm Sorting techniques in high Sperm DNA Fragmentation Index Jindal IVF & Sant Memorial Nursing Home Chandigarh India

2. Introduction • Sperm DNA damage can be defined as any chemical change in the normal structure of the DNA. • Sperm DNA fragmentation (sDF) is one of the most common disturbances affecting the genetic material in the form of single or double strand breaks.

3. DNA damage Mechanism

4. Indications of Sperm DNA Fragmentation Testing • Unexplained or persistent infertility • Failure to conceive after 5-6 intra uterine insemination (IUI) cycles despite good count and motility • Low fertilization rates or poor embryo quality in IVF cycles • Recurrent miscarriage • Prolonged stay in an environment that exposes to reproductive toxins • Abnormal semen analysis • Advancing male age (>45 years) • Smokers

5. Types Sperm DNA Fragmentation Testing

6. Testing Cut off Ranges

7. Prognostic value of sperm DNA damage in predicting clinical pregnancy L. Simon, L. Liu, K. Murphy, S. Ge, J. Hotaling, K.I. Aston, B.Emery,and D.T. Carrell: Human Reproduction, Vol.29, No.5 pp. 904–917, 2014

8. Best method for Sperm DNA Fragmentation

9. Diagnostic cut-off point at JISNH

10. Objectives of Sperm sorting To maximize the chances of fertilization by obtaining SPERMATOZOA with the highest potential for fertilization from semen sample by 1) Removal of prostaglandins. 2) Removal of pathogens. 3) Removal of antibodies. 4) Removal of debris & dead spermatozoa. It cannot increase or improve the basic sperm quality or number

11. Final Outcome The final outcome is to select viable, motile and morphologically intact sperm by significantly reducing the percentage of spermatozoa with nuclear abnormalities and DNA damage.

12. Sperm Sorting • For IUI • For IVF • For ICSI

13. Selection of Technique Ideal technique Proper technique Since none of the methods available meets all these requirements, a variety of sperm separation techniques is mandatory in clinical practice to obtain an optimal yield of functionally competent spermatozoa for insemination purposes. Depending on the ejaculate quality, these methods have different efficiency and areas of use • Quick, easy and cost-effective. • Isolate as much motile sperm as possible. • No sperm damage or physiological alterations. • Eliminate dead spermatozoa and non sperm elements, toxic or bioactive substances like decapacitation factors or ROS. • Allow processing of larger volumes of ejaculates

14. Functional & Molecular Assay • Capacitationtests • Zona-freehamster penetrationassay • Membrane integrity tests • Antisperm antibodies • Vital staining • Biochemicalanalysis • Peroxidasestaining • Sperm ubuquitintag immunoassay(SUTI) • Semen culture • Hypo-osmotic swellingtest • Sperm penetration assay Hemizonaassay • Creatinkinase • Reactive Oxygen Species(ROS) • CASA

15. Types of Techniques

16. Progress so far in widely adopted in clinical practice A. Simple Washing C. Density Gradient B. Direct- Swim up

17. Swim-up Based on motility. Used for IUI Only for good semen sample Sperm recovery is less

18. Density Gradient Based on adherence due to density. Used for IUI/IVF/ICSI Used for samples with pus cells etc High recovery of sperm

19. DNA Fragmentation (%) Aldo Volpes, Francesca Sammartano, Simona Rizzari, Salvatore Gullo, Angelo Marino, Adolfo Allegra. Journal of Assisted Reproduction and Genetics; Gamete Biology DOI 10.1007/s10815-016-0696-2, First online: 16 March 2016

20. Traditional sperm parameters & ICSI Success of ICSI independent of traditional spermparameters Hum Reprod,1995

21. New sperm parameters & ICSI New sperm parameters are associated with ICSIoutcome Fertil Steril,2008

22. Advanced Techniques

23. MSOME(Motile Sperm Organelle Morph. Exam) • Examination performed in real time on living Sperm. • Inverted light microscope • Equipped with high-power Nomarski optics instead of Hoffman Modulation Contrast • Enhanced by digital imaging to achieve a magnification up to 6300. • More accurate examination of spermatozoa

24. Equipment for MSOME ICSI 2-400 XHoffman Palermo et al.,1992 MSOME >6600XNomarski Bartoov et al.,

25. Motile Sperm Organelle Morphology Examination -MSOME

26. Criteria Motile sperm organellar morphology examination(MSOME) Bartoov et al., 2002 Based on ultrastructural studies of acrosome, postacrosomal lamina, neck, mitocondria, tail and nucleus Glezermanand Bartoov, 1993; Bartoov et al.,1994 No vacuolesor less than 4% of thenuclear surface Head shape normal= Oval, smooth and symmetrical, size 4.75 ± 0.28 μm (length)to 3.28 ± 0.20 μm(width) Chromatin content normal= no vacuolesor the vacuoles occupy < 4% of the nuclear surface (0.78 ± 0.18μm)

27. VACUOLES • Association between vacuoles and sperm DNA fragmentation (Garolla et al., 2008; Franco et al., 2008; Berkovitz et al., 2005; Oliveira et al.,2010) • Association between vacuoles andaneuploidy (Garolla et al.,2008) • Lower risk of sex chromosome abnormalities in IMSI vs. IVF (OR 0.57, CI 0.37-0.90) Figueira et al.,2011

28. VacuolesFertilization & embryodevelopment • Vacuoles associated with lower fertilizationrates (Cassuto et al., 2009; Franco et al.,2008) • Correlation between presence and size of vacuoles and cleavage stageembryodevelopment (Berkovitz et al., 2005;2006a,b) • No correlation between presence and size of vacuoles and cleavage stage embryo development (Hazout et al., 2006; Antinori et al., 2009; Mauri et al., 2010; Balaban et al.,2011) • Correlation between presence and size of vacuolesandblastocyst development Vanderzwalmenet al.,2008

29. IMSI vs.ICSIImplantation andpregnancy • Implantation and pregnancy results higher in IMSI vs ICSI group Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al 2006; Berkovitz et al., 2006a,2006b; • Abortion rates lower in IMSI vs. ICSI group Bartoov et al., 2003; Berkovitz et al., 2005; Hazout et al 2006; Berkovitz et al., 2006a,2006b

30. Magnetic Activated Cell Sorting

31. What is MACS? • Magnetic activated cell sorting of human spermatozoa • An advanced sperm preparation technique working on the principle to separate sperm cells with apoptotic features .

32. Apoptosis • Apoptosis is the programmed cell death that occurs because of the DNA fragmentation which is seen in the sperm of infertile men. • Sperm cells with apoptotic features can remain normally shaped • They may still be able to fertilize an oocyte.

33. MACS: Technique Semen sample is mixed with supraparamagnetic beads conjugated to highly specific antibodies to annexin-V are incubated at room temperature for 15 minutes . The mixture is loaded on top of separation column which is placed in the magnetic field

34. MACS Pros Cons MACS need to be used in conjunction with other technique such as DGC to remove seminal plasma • Rapid, convenient, non invasive • Acts at molecular level • Only technique that separates apoptotic sperm • Provides optimal purity and reliable and consistent results • Optimise cryopreservation thawing outcomes

35. Sperm selection based on surface charge Electrophoretic method Zeta potential

36. Principle • Mature spermatozoa carry an electronegative surface charge, which is attributed to sialic acid residues including CD52 found on sperm plasma membrane. • CD52 is acquired during epididymal maturation. Its presence indicates normal sperm morphology and capacitation; therefore, it can be considered as an indication of sperm maturity and quality. Electrophoresis technique • External electrical current is applied and mature negatively charged spermatozoa moves towards positive electrode

38. Hyaluronan Binding Theory • Mature sperm have completed the plasma membrane remodeling and have receptors for and can bind to hyaluronic acid (HA) • Immature sperm have not developed receptors for hyaluronic acid and will not bind to HA

39. Hyaluronan Binding Theory A sperms ability to bind to HA correlates to: • Kruger Strict Morphology • Cellular maturity, • Less rates of chromosomal aneuploidy, • Less rates of DNA fragmentation, • Increased chromatin integrity • Normal head morphology = better fertilizing potential

40. What is Hyaluronan ? • Hyaluronan (Hyaluronic acid, HA): • Is an anionic, non-sulfated glycosaminoglycan • Is the major component of the Cumulus Oophorus complex surrounding the human oocyte. • Provides viscoelastic properties for the cumulus structures • HA assists the binding of the cumulus cells together and importantly actsto activate the sperm whilst in the cumulus.

41. HB Assay – HyaluronanBinding Assay Diagnostic tool • - Sperm sample evaluation in minutes • Is an important diagnostic tool used in the analysis of semen. • In a matter of minutes it provides an answer to the proportion of mature sperm in the sample (HBA score). • ONLY mature sperm with developed HA receptors bind • Low HBA score ≤65% : • Decreased quality of sperm sample = evaluate further treatment (IUI/IVF/ICSI) • Decreased likelihood of selecting best sperm for ICSI using morphology evaluation only

42. HB Assay – HyaluronanBinding Assay Diagnostic tool Designed with two duplicate chambers coated with HA

43. Mature spermatozoashow a reduction of more than five fold in aneupliody rate than immature ones.

48. PICSIdish • ICSI dish with hyaluronan coated dots for sperm selection • Bound sperm = Mature sperm with high DNA integrity • Benefit with PICSI increase as HBA score decrease (low binder samples) Important: Could reduce number of unexplained failures by preventing injection of “good-looking” (but immature) sperm

49. PICSI Pros Cons Sperm are selected individually i.e. more demanding and longer process . Used media can affect sperm Only for motile sperm • Physiological/Natural process of selecting sperm

50. Sperm Slow • Semi viscous medium containing Hyaluronic acid • Natural alternative to PVP = ONLY slows sperm with HA receptors (e.g. Mature sperm with high DNA integrity) • PVP may induces nuclear damage and chromosomal aberration. • PVP injected into the egg along with the sperm cannot diffuse out or be broken down intracellular & remain in the developing embryo for a prolonged period, where it is likely to impede embryo development and pregnancy.