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Laser Use in ART

Laser Use in ART. Aygul Demirol, M.D Medical Director, CLINIC Women Health, Infertility and IVF Center Ankara/Turkey. Laser Use in ART. Assisted Hatching ( Strohmer,1992;Antinori,1996;Montoidis,2001, Hisieh 2002, Primi 2004)

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Laser Use in ART

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  1. Laser Use in ART Aygul Demirol, M.D Medical Director, CLINIC Women Health, Infertility and IVF Center Ankara/Turkey

  2. Laser Use in ART • Assisted Hatching (Strohmer,1992;Antinori,1996;Montoidis,2001, Hisieh 2002, Primi 2004) • PGD –Microdissection of ZP with a laser system simlifies subsequent polar body biopsy or removal of blastomers(Veiga,1997;Boada,1998;Montag,1998, Rienzi 2004, Nagy 2005) • Defragmantation • Necrotic blastomere removal • Sperm immobilization (Tadir,1989, Ebner 2001) • Laser Assisted ICSI( Nagy,2001;Abdelmassih,2002, Rienzi 2004, Demirol 2006)

  3. Assisted Hatching (AH) The introduction of assisted hatching by J. Cohen (HR 1990) offered an additional tool for assisting implantation in patients undergoing ART

  4. Different Techniques for Assisted Hatching • Mechanical • Acid Tyrode • Thinning of zona by protease • Laser

  5. Laser Use in Assisted Hatching • Mechanical and chemical techniques might have minimal adverse effects on embryos (injury to the embryos??) • Rapid, no-touch microdrilling, efficient, precise, safer and chemical free

  6. Laser Assisted Hatching • Routine Laser AH results significantly increased clinical pregnancy rates (Ali et al.,J Assist Rep Genet,2003) • Selective assisted hacthing using four different tecniques yields similar implantation and pregnancy rates (Balaban et al.,HR,2002) • Laser AH did not improve the pregnancy rate and implantation rate of frozen thawed embryo transfer cycles ( Ng et al. HR,2005)

  7. Laser Assisted Hatching • LA hatching of embryos is more effective than the chemical method in enhancing the IR and PR of women with advancing age. 1.48 non-touch diode laser is easier, faster and safe (Montag et al., CMJ;1999,Hsieh et al.,FS;2002) • No increase in the incidence of chromosomal aberrations and congenital abnormalities in 134 chidren born after using this technique (Kanyo and Konc,Eur J Ob Gyn,2003) • Clinical pregnancy rates arising from quarter LAH is higher in comparision with partial and total LAH (Mantoudis;HR,2001)

  8. Laser Assisted Hatching vs Chemical Assisted Hatching: prospective study • 601 embryos from 141 patients aged ≥ 38 yrs, first IVF cycle • 85 patients in laser group and 56 patients in chemical group • Laser assisted hatching using 1.48 µm non-contact diado laser • Chemical assisted hatching using acid Tyrode’s solution Hsieh et al., 2002, Fertil Steril 2002; 78: 179-182

  9. Hsieh et al., 2002, Fertil Steril 2002; 78: 179-182

  10. Balaban et al. 2002, Hum Reprod; 17: 1239-1243

  11. H. Sallam et al, 2003, meta analysis

  12. F&S 2007

  13. F&S 2007

  14. Different types of laser AH Comparison of quarter, partial and total laser AH in selected infertility patients Mantoudis 2001, HR

  15. Mantoudis et al, 2001 Hum Reprod; 16: 2182-2186

  16. Mantoudis et al, 2001 Hum Reprod; 16: 2182-2186

  17. Cochrane Database Syst Rev. 2006 Jan 25;(1):CD001894. Clinical pregnancy rate per woman Twenty-four trials reported clinical pregnancy data, including 954 clinical pregnancies in 2889 women. The odds ratio for clinical pregnancy per woman randomised was 1.29 (95% CI 1.10 to 1.52), significantly in favour of assisted hatching (p<0.001)

  18. Cochrane Database Syst Rev. 2006 Jan 25;(1):CD001894 Live birth rate • Few trials reported live birth data, with data available from only six of the 23 trials. Overall, 163 live birth events were reported (i.e. not includng individual births from multiples) There was no evidence of a significant difference between the odds of a live birth in women who underwent assisted hatching compared with those in the control group (random effects odds ratio 1.19, 95% CI 0.81 to 73)

  19. F&S 2007

  20. F&S 2007

  21. Laser Assisted AHA

  22. Laser Assisted Zona Drilling for PGD • Procedure makes polar body biopsy more accurate and effective (Montag et al.,FS;1998) • Sucessful outcomes demonstrate the efficacy and safety of the laser assisted embryo biopy to facilitate PGD (Han et al.,FS;2003) • The use of laser in cases of PGD is an easier procedure and results more intact blastomers in comparison with using acid Tyrode medium.Since similar pregnancy rates are obtained ,it is adventageous to use laser for zona drilling (Joris et al.,HR;2003)

  23. Comparison of the Results of Human Embryo Biopsy and Outcome of PGD After Zona Drilling Using Acid Tyrode Medium or a Laser • Zona pellucida opennig with AT or Laser for PGD;Results of embryo biopsy and PGD in two periods compared • Fewer blastocyts were intact with ATD 95.2% vs 98.3% • Ongoing PR and ongoing IR did not differ • The use of LZD in cases of PGD is an easier procedure and results in more intact blastomeres.Since similar pregnancy rates obtained,it is adventageous to use laser • Further follow up is necessary to prove the safety of the procedure (Joris et al.,HR2003)

  24. Laser PGD

  25. Laser DFRG

  26. Laser Sperm Immobilizasyon

  27. Laser assisted sperm immobilization prior to ICSI • Potentially useful alternative to the conventional mechanical approach • Some studies showed the effectiveness of this method (Montag 2000, Ebner 2001) • Making laboratory work simpler, quicker without lowering fertilization

  28. The application of a single laser shot to the far end of the sperm tail causes a curling of the sperm tail only in viable sperm, similar to the reaction observed in the hypo-osmotic swelling test.

  29. Removal of necrotic blastomeres from partially damaged frozen-thawed embryos before transfer increased rates of pregnancy (45.7% vs. 17.1%), ongoing pregnancy (40.0% vs. 11.4%) and ongoing implantation (16.2% vs. 4.3%) compared with the control group, in which necrotic blastomeres were not removed.

  30. Effect of ZP openning on clinical outcome of ART in patients with advanced age, RIF or frozen/thaw • Prospective randomized trial • IVF OR ICSI • Advanced age (≥37 yr) (n=410) • RIF (≥2) (n=796) • Frozen/thawed ET (n=180) Valojerdi et al, F&S in press

  31. Advanced age PR (LAH : 15.1%) PR (non LAH : 21%) IR (LAH : 6.5%) IR (non LAH : 9.1%) RIF PR (LAH : 27.1%) PR (non LAH : 26.9%) IR (LAH : 11.6%) IR (non LAH : 12.9%) Results with advanced age and RIF: NO DIFFERENCE Valojerdi et al, F&S in press

  32. Results with frozen/thawed ET: • PR (LAH 31.2% & non LAH 11.1% p 0.001) • IR (LAH 12.8% & non LAH 4.2% p 0.000) Valojerdi et al, F&S in press

  33. Laser assisted ICSINEW ECHNIQUE

  34. Conventional ICSI • Is highly efficient in achieving high normal fertilization • May be associated with degeneration of oocytes - Technical conditions - Oolemma fragility(Nagy,1995;Palermo,1996) - Difficulty in oolemma breakage(Vanderzwalmen,1996) - Resistant zona pellucida ( Nagy,1995)

  35. ICSI • Especially if few oocytes retrieved • High degeneration rate • Poor embryo quality mayeffect the pregnancy and implantation rates NEGATIVELY!

  36. ICSI • Severe mechanical stress or difficulty in penetration during ICSI may damage or disorganize the cytoskelon of MII oocytes (Dumoulin,2001;Ebner,2001)

  37. Types Oolemma Membrane Breakge • Sudden breakage Type A1: Breakage occurs at the beginning of the ICSI Type A2: Breakage occurs more deeply in the ooplasm • Normal breakage Type B • Difficult Breakage Type C,D and Ewhen strong aspiration and/or reposition of injection needle is required to break the membrane

  38. Difficult Breakage Pattern • May serve a marker for a dysfunction of zona pellucida (Ebner et al.,HR,2002)

  39. Laser Systems • Contact type- ER:YAG (Obruca,1997) • Noncontact - Ho:YSGG UV Laser (Liow,1996) • 1480 mm diode laser (Germond,1996;Rink,1996;Montag,1998;Blake,2001)

  40. 1480 mm Diode Laser • Works without physically touching the cells • Has no detectable detrimental effects on living cells especially used with short pulse duration less than 5 ms and laser power 100 mW (Catzimeletiou,2001;Nagy,2001) • Is easy to handle ;No mutagenic effect • The distance between perivitelline space and oolemma should be maximum point of laser drilling • Innermost layer of ZP kept intact

  41. Laser Assisted ICSI (LA-ICSI) • Using laser beam generated by a 1480 mm diode laser • A channel with smaller diameter (5-6 mmic) was drilled • Three to five pulses of 10-15 msec (depending to the charesteristic of ZP) • The injection pipette is introduced through this channel and microinjection is performed as usual

  42. Laser-assisted ICSI • Drilling a microhole on the ZP of the oocytes by laser beam just prior to ICSI • Penetration of the microneedle without any trauma

  43. EASY and DIFFICULT ICSI

  44. Laser Assisted ICSI

  45. Repeated ICSI failure caused by oocyte degeneration In LA-ICSI cycle, Survival of 8 oocytes out of 13 injected, normal fertilization in 5 oocytes Clinical pregnancy was established Rienzi et al, 2001 Fertil Steril Two previous failed IVF cycles with high degeneration of oocytes In LA-ICSI cycle, 11 MII ocytes of same patient, 5 oocytes with conventional ICSI, 6 oocytes with LA-ICSI High fertilization and better embryo quality resulted in pregnancy with LA-ICSI Nagy et al, 2001 RBM Online Case reports of LA-ICSI

  46. Randomized study related to LA-ICSI • 32 patients (32 cycles), previous failed ICSI cycles with high degeneration of oocytes • Oocytes of the same patients randomly divided • LA-ICSI and conventional ICSI(C-ICSI) • 201 oocytes in LA-ICSI group and 137 oocytes in C-ICSI Abdelmassih et al, 2002 Hum Reprod; 17: 2694-2699

  47. Randomized study related to LA-ICSIResults • Survival rates significantly higher in LA-ICSI • Sudden breakage of the oocytes membrane significantly low in LA-ICSI • Normal fertilization rate not different between the groups • The parcentage of excellent quality embryos significantly higher in LA-ICSI Abdelmassih et al, 2002 Hum Reprod; 17: 2694-2699

  48. LA-ICSI • Complete or incomplete degeneration of oocytes may impair the changes of pregnancy by reducing the number of embryos (Lui et al.,1995) • More sensitive oocytes not only degenerate but the developing embryos may be poorer quality as a result of the sublethal demaging force of the traumatic injection • This may be responsible for the observation that embryo quality was poorer after C-ICSI and better after LA-ICSI (Nagy et al.,2001; Abdelmassih,2002)

  49. Laser Assisted ICSI can be appliedto • Oocytes showing an increased elasticity of the oolemma (Rienzi,2001;Abdelmassih,2002) • In oocytes showing an inherent fragility of the membrane (Abdelmassih,2002;Nagy.2002) • To rescue oocytes after failed fertilization with conventional IVF( Eroğlu,2002) • Oocytes showing normal membrane response (Nagy, 2004;Moser,2004)

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