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Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
Alpesh optimising icsi (including imsi)
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Alpesh optimising icsi (including imsi)

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  • 1. Optimising ICSI (Including IMSI) Alpesh Doshi Head of Embryology Research Instruments Workshop Jordan March 2012 1
  • 2. Indications for ICSI • Male factor – – – – – – – – – – • • Oligozoospermia (<10 million/ml) Asthenozoospermia (<40% progressive motility) Teratozoospermia (<3% normal forms) Antisperm Antibodies (>35% IgA or IgG) Globozoospermia Ejaculatory disorders (retrograde, electroejaculation) Congenital absence of the Vas deferens Obstruction of ejaculatory ducts Failed Vasectomy reversal Non obstructive/Obstructive Azoospermia Poor/ No Fertilisation after IVF (Zona receptor binding) PGD-Single gene defects –Paternal Contamination 2
  • 3. Equipment • Micromanipulators • AntiVibration tables/ Platforms • Microtools 3
  • 4.  Equipment  Location 4
  • 5.  Equipment  Micro Tools  Spike/ Non spike, Angle, Internal/ Outer diameter, MEA test, CE marked, packaging, Cost.  Avoid making your own ! 5
  • 6.  Equipment Select carefully Consider: •Ease of use •Reliability •Service available locally 6
  • 7. Sperm preparation techniques • Sperm Preparation Techniques need to be: -Swim up, Density gradients (300g force) • PESA- Wash, Mini density gradient • Testicular (TESE) sperm preparationMilking/washing, Mini DG (Rare) - ELB, Collagenase txt, Pentoxifylline (if necc) • Frozen sperm- Long Incubation post thaw showed High DNA damage (Dalzell et al 2004) incubation time post thaw showed High DNA damage (Dalzell et al 2004) 7
  • 8. • Sperm Selection – At 400X – PVP used to slow down movement – Normal Sperm selected – Morphology of head, motion pattern and light refraction are considered – Immotile and poorly progressive sperm have centrosomal damage (Sathananthan and Trounson 2000) 8
  • 9. • Sperm Selection – For PESA/TESA and severe oligospermia • • • • At 400X Use long drops of the sperm suspension (washed well) Select sperm and deposit into the PVP holding droplet. How long should this take? 9
  • 10.  The Procedure - Some considerations   Searching for testicular sperm >1000 NOA TESE/ ICSI Cycles Fert Rate Pregnancy Rate 30mins-1hr 54.2% 41.2% 1hr-2hr 46.3% 36.6% 2hr-3hr 28.0% 26.8% >3hrs 25.4% 21.2% D Monahan et al, Oral presentation ESHRE 2011 10 10
  • 11. Pentoxifylline/ Theophylline .... • Phosphodiesterase inhibitor/ Caffeine derivative. • Mangoli et al 2011 (Fertil steril) : Higher FR and CPR in Pentox group compares to HOS • DeMendosa et al 2000 (Fertil Steril) • Kovacic et al 2006 (J Androl) • Griveau et al 2006 (RBM online) • Gioretti et al 2005(RBM online) 11
  • 12. CRGH Protocol • Pentoxifylline/theophylline (Gynemed- Germany) working solution made fresh (1mg/ml) • Warm to 37°C. • Add 1 :1 ratio of sperm suspension/ Pentox • Isolate motile and morphologically normal spermatozoa into PVP drop. • Wash in PVP, immobilise and Inject. • Do not exceed 10 mins in pentox solution. 12
  • 13. Video (Pentox) 13
  • 14.  The Procedure - Some considerations   HEPES or culture media? -Time consideration, pH, toxicity- balance! • Morgia et al 2006 (Fertil Steril)- sign higher triploidy, damage rate with oocytes exposed to hepes. Sign lower good quality embryos, lower implantation and pregnancy rate in hepes group. - Exposure to PVP • Hlinka et al 1998 (Hum Reprod) sign higher fert rate with no PVP used -Immobilisation method   No advantage of aggressive swipe techniques (A Velaers et al ESHRE 2011) One gentle swipe “accurate set up of micro tools is essential” 14
  • 15. Polar Body positioning • Anifandis et al (2010)- Reprod sci. - Sign higher fertilisation rate, good quality embryos and pregnancy rate with oocytes injected with PB at 11 o’clock compared to 6, 7 or 12 o’clock • Woodward et al (2008) RBM Online - Highest frequency of normal fertilised oocytes and good quality embryos with injection in/ near the plane of spindle ie at 3, 4, 8 and 9 o’clock 15
  • 16. At CRGH: Post hCG... • • • • • • • • • Egg collection- 37 hrs Sperm collection- 37 hrs Sperm preparation -37.5 hrs Sperm incubation - up to 40-41 hrs(min 1 hr inc) Denudation -40 hrs (39 if large egg no/TESE sperm) ICSI- 41 hrs Large egg no’s or TESE sperm – start ICSI at 40 hrs ICSI complete by 41.5- 42 hrs Fert check 16-18 hrs post ICSI 16
  • 17.  The Procedure - Some considerations  Timing of ICSI ( Dozortsey et al Fertil Steril 2004)     Fert rates increase with time elapsed post hCG with optimal at >41 hrs. Highest implantation rates achieved at 37-41 hours post hCG Lower implantation rates achieved at <37hrs or >41hrs are due to metabolic incompetence either metabolic immaturity or post maturity. “Don’t leave your ICSI cases till the end of the day” 17
  • 18. The Oocyte.... 18
  • 19. Oocyte preparation • VEC 36-37 hrs post hCG • Preincubation of 2-4 hrs resulted in improved maturation of oocytes, fertilisation and embryo quality.(Isiklar et al 2004) • Denudation using Hyaluronidase (10-80IU/ml) – Higher conc and exposure time induces parthenogenesis (Van de velde et al 1997) 19
  • 20. Microscopic Evaluation of morphology and maturity • Evaluated under x400 mag • 10-12% of oocytes immature (GV, M1). Score and separate at denudation • In vitro matured oocytes. Very poor fert & preg rates (De Vos 1999, Nagy 1996). High chromosomal abnormailities (Picton H. personal communication.) • Metaphase II oocytes graded according to cytoplasmic & polar body integrity.(Xia 1997, Serhal 1998) 20
  • 21. Temperature and the spindle A DROP IN TEMPERATURE CAN EQUATE TO A DEPOLYMERIZED SPINDLE –Temperature fluctuations can induce de polymerisation and hence non disjunction of chromosomes leading to aneuploidies (Wang et al 2001) 21
  • 22. Temperature and spindle • Pickering et al (1990) Fert Steril. – Microtubule disorganisation, reduction in spindle size, complete lack of spindle seen in all oocytes when exposure time was 30 mins to rtp. • Almeida & Bolton (1995) Zygote – 77% of oocytes had spindle disruption when exposed for 2 mins at rtp. Chromosomal dispersal in 50%. Effect irreversable when time exceeded 10 mins. • Wang et al (2001) Human Reprod. – Spindle depolymerisation by 5 mins when exposed to to rtp. 22
  • 23. Individual testing in different labs based on size of drops, type and diameter of dishes and room temp. 23
  • 24. Injection.. • Aspiration volume of cytoplasm into pipette: -Dumoulin et al 2001- sign reduced blast rate with >6pl of cytoplasm aspirated -Hiraoka et al (2011) ESHRE oral pres: Sign higher Fertilisation rate in oocytes with less cytoplasm aspirated. 24
  • 25. It’s not getting the sperm in, it’s getting the right sperm in that matters 25
  • 26. • Finding the best sperm “Physiologic ICSI”: Hyaluronic acid (HA) favors selection of spermatozoa without DNA fragmentation and with normal nucleus, resulting in improvement of embryo quality Lodovico Parmegiani et al Fertil Steril 2010 26
  • 27. IMSI
  • 28. 28
  • 29. • Finding the best sperm - IMSI IMSI Workstation Digital Camera Objective 29
  • 30. • Finding the best sperm - IMSI Gris Reproduccion, Barcelona 30
  • 31. IMSI grading GRADE I No vacuoles GRADE II < 2 vacuoles I GRADE III > 2 vacuoles or at least one large vacuole GRADE IV II Large vacuole and abnormal head shapes or other abnormalities IV III Sperm was selected using Vanderzwalmen et al., (2008) grading system 31 31
  • 32. 32
  • 33. Meta analysis- IMSI Vs ICSI – 37 studies in literature – Only 3 were comparative or randomised. – Outcomes: Fertilisation, implantation, Pregnancy and Miscarriage rates. Souza Setti et al 2010 RBM Online 33
  • 34. Souza Setti et al 2010 RBM Online 34
  • 35. Meta-analysis showed: Outcome ICSI IMSI Conclusion Fertilisation Rate 76.7% 75.7% NS Top Quality Embryos (2 studies) 27.7% 41.2% Statistically Significant Implantation Rate 10.5% 21.9% Statistically Significant Pregnancy Rate 26.6% 47.6% Statistically Significant Miscarriage Rate 29% 14.7% Statistically Significant Souza Setti et al 2010 RBM Online
  • 36. Clinical Outcome of IMSI: A prospective Randomised Study Balaban et al (2011)- RBM Online Unselected Population -No Significant Difference seen in outcome measures. Severe Male factor Group - Significantly higher Implantation rates. 36
  • 37. 37
  • 38. 38
  • 39. 39
  • 40. Study Significant Findings (MSOME & IMSI vs. ICSI) Souza Setti et al, 2010 Significantly higher pregnancy and implantation and significantly lower miscarriage rate in IMSI group Figueira et al, 2010 Significantly lower aneuploidy rate in IMSI group Wilding et al, 2010 64.8% of sperm selected in ICSI had significant DNA fragmentation. Embryo quality, pregnancy and implantation rate higher in IMSI group Monquat et al, 2010 Swim up sperm have less nuclear vacuolation than DGS Vanderzwalmen et al, 2008 Presence of nuclear vacuoles reduces PR, higher pregnancy rate in IMSI group. Proposed sperm grading scheme Antinori et al 2008 +ve correlation for pregnancy and miscarriage in OAT group Bartoov et al, 2002 Significantly higher pregnancy rate in IMSI group Bartoov et al, 2002 Describe MSOME. Sperm with vacuole occupying >4% of nuclear area abnormal and not used for injection Bartoov, 2001 First reported IMSI. Magnification x6000. 40
  • 41. Future.....
  • 42. SEM Nomarski Polarized Native Native Fixed TEM Fixed • Nasum External Information Courtesy: Dr Sergei Yakovenko, Altravita Internal 42
  • 43. Future Aim to understand how the methods of external spermatozoa observation (such as Nomarsky, Hoffman, SEM) reflect the internal structure of spermatozoa (NASUM and TEM). Courtesy: Dr Sergei Yakovenko, Altrvita
  • 44. Nasum (Native assesment of sperm ultra morphology) • Simultaneous use of Nomarsky and Hoffmans contrast • Resolution increased by circular polarised light • Additional lenses give a total magnification of x 20000 (including video zoom) Courtesy: Dr Sergei Yakovenko, Altrvita
  • 45. 102 ,99 Courtesy: Dr Sergei Yakovenko, Altrvita
  • 46. Thank you Alpesh.doshi@uclh.nhs.uk

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