1. Laser Use in ART 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) 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) 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 Defragmantation Necrotic blastomere removal Necrotic blastomere removal Sperm immobilization (Tadir,1989, Ebner 2001) Sperm immobilization (Tadir,1989, Ebner 2001) Laser Assisted ICSI ( Nagy,2001;Abdelmassih,2002, Rienzi 2004, Demirol 2006) Laser Assisted ICSI ( Nagy,2001;Abdelmassih,2002, Rienzi 2004, Demirol 2006)

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

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

5. Laser Use in Assisted Hatching Mechanical and chemical techniques might have minimal adverse effects on embryos (injury to the embryos??) 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 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) 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) 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) 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 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) (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) 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) 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 601 embryos from 141 patients aged ≥ 38 yrs, first IVF cycle 85 patients in laser group and 56 patients in chemical group 85 patients in laser group and 56 patients in chemical group Laser assisted hatching using 1.48 µm non- contact diado laser Laser assisted hatching using 1.48 µm non- contact diado laser Chemical assisted hatching using acid Tyrode’s solution Chemical assisted hatching using acid Tyrode’s solution Hsieh et al., 2002, Fertil Steril 2002; 78: 179-182

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

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

13. F&S 2007

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

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

18. 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)

19. 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) Cochrane Database Syst Rev. 2006 Jan 25;(1):CD001894 Live birth rate

20. F&S 2007

22. Laser Assisted AHA

23. Laser Assisted Zona Drilling for PGD Procedure makes polar body biopsy more accurate and effective (Montag et al.,FS;1998) 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) 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 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) (Joris et al.,HR;2003)

24. 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 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% Fewer blastocyts were intact with ATD 95.2% vs 98.3% Ongoing PR and ongoing IR did not differ 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 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) Further follow up is necessary to prove the safety of the procedure (Joris et al.,HR2003)

25. Laser PGD

26. Laser DFRG

27. Laser Sperm Immobilizasyon

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

29. 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.

30. 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.

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

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

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

34. Laser assisted ICSI NEW ECHNIQUE

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

36. ICSI Especially if few oocytes retrieved Especially if few oocytes retrieved High degeneration rate High degeneration rate Poor embryo quality Poor embryo quality may effect the pregnancy and implantation rates may effect the pregnancy and implantation rates NEGATIVELY! NEGATIVELY!

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

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

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

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

41. 1480 mm Diode Laser Works without physically touching the cells 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) 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 Is easy to handle ;No mutagenic effect The distance between perivitelline space and oolemma should be maximum point of laser drilling The distance between perivitelline space and oolemma should be maximum point of laser drilling Innermost layer of ZP kept intact Innermost layer of ZP kept intact

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

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

44. EASY and DIFFICULT ICSI

45. Laser Assisted ICSI

46. Case reports of LA-ICSI Repeated ICSI failure caused by oocyte degeneration Repeated ICSI failure caused by oocyte degeneration In LA-ICSI cycle, Survival of 8 oocytes out of 13 injected, normal fertilization in 5 oocytes In LA-ICSI cycle, Survival of 8 oocytes out of 13 injected, normal fertilization in 5 oocytes Clinical pregnancy was established 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

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

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

49. LA-ICSI Complete or incomplete degeneration of oocytes may impair the changes of pregnancy by reducing the number of embryos (Lui et al.,1995) 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 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 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) (Nagy et al.,2001; Abdelmassih,2002)

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

51. Comparison of the laser-assisted ICSI and conventional ICSI results in recurrent ICSI failure patients with few oocytes are available Aygul Demirol, Tamer Sari and Timur Gurgan. CLINIC IVF Center, Ankara-TURKEY ESHRE 2003, oral presentation

52. Use of laser-assisted (ICSI) in patients with a history of poor ICSI outcome and limited metaphase II oocytes A.Demirol, M. Benkhalifa, T. Sari, T. Gurgan Fertil Steril 2006, 86: 256

53. Objective To investigate if laser assisted ICSI (LA-ICSI) improves the cycle outcome in recurrent conventional ICSI failure patients which few oocytes are available To investigate if laser assisted ICSI (LA-ICSI) improves the cycle outcome in recurrent conventional ICSI failure patients which few oocytes are available For evaluation of the outcome, we compared the results of LA-ICSI and C-ICSI on the sibling oocytes For evaluation of the outcome, we compared the results of LA-ICSI and C-ICSI on the sibling oocytes Demirol et al, F&S 2006

54. Material and methods Forty patients (forty ICSI cyles) Forty patients (forty ICSI cyles) Four or less M II oocytes were retrieved Four or less M II oocytes were retrieved Two or more failed ICSI cycles Two or more failed ICSI cycles Mean age 38.6 yrs Mean age 38.6 yrs Demirol et al, F&S 2006

55. Material and methods M II oocytes from the same patients were randomized into two groups M II oocytes from the same patients were randomized into two groups Group I: laser assisted ICSI (L-ICSI) and group II: conventional ICSI (C-ICSI) Group I: laser assisted ICSI (L-ICSI) and group II: conventional ICSI (C-ICSI) Oocytes were retrieved by transvaginal USG guided 36 hours after HCG administration Oocytes were retrieved by transvaginal USG guided 36 hours after HCG administration Stored in incubator at 37 0 C with 5% CO 2 for 2-4 hours Stored in incubator at 37 0 C with 5% CO 2 for 2-4 hours Demirol et al, F&S 2006

56. To ensure the maximum safety The greatest care was exercised during the procedure The greatest care was exercised during the procedure The distance between the perivitelline space and oolemma was the maximum at the point of laser drilling The distance between the perivitelline space and oolemma was the maximum at the point of laser drilling The innermost layer of the zona pellucida(ZP) (~0.5µm) was kept intact The innermost layer of the zona pellucida(ZP) (~0.5µm) was kept intact very short pulse duration (<2 ms) was applied very short pulse duration (<2 ms) was applied No visible sign of oolemma reaction was observed No visible sign of oolemma reaction was observed Demirol et al, F&S, 2006

57. LA-ICSI was performed in the following manner-I Oocytes and spermatozoa were placed into an injection dish as usual Oocytes and spermatozoa were placed into an injection dish as usual Single and immobilized spermatozoon was aspirated into the injection pipette Single and immobilized spermatozoon was aspirated into the injection pipette Prior to microinjection, the oocyte to be injected was secured on the holding pipette so that the maximum possible distance was present between the inner surface of the ZP and oolemma(perivitelline space)at the 3 o’clock position (where the injection needle would penetrate into the oocyte) Prior to microinjection, the oocyte to be injected was secured on the holding pipette so that the maximum possible distance was present between the inner surface of the ZP and oolemma(perivitelline space)at the 3 o’clock position (where the injection needle would penetrate into the oocyte) Demirol et al, F&S 2006

58. LA-ICSI was performed in the following manner-II Using a laser beam generated by a 1480 nm diado laser (Saturn 2 Laser System, Research Instruments, UK) Using a laser beam generated by a 1480 nm diado laser (Saturn 2 Laser System, Research Instruments, UK) A channel with small diameter (5-6 µm) was drilled with 3-5 low energy pulses, always using <2 ms of pulse duration A channel with small diameter (5-6 µm) was drilled with 3-5 low energy pulses, always using <2 ms of pulse duration The injection pipette was introduced through this channel and ICSI was performed as usual The injection pipette was introduced through this channel and ICSI was performed as usual Demirol et al, F&S, 2006

59. Material and methods Group I: 64 oocytes, laser-assisted ICSI (L-ICSI) Group I: 64 oocytes, laser-assisted ICSI (L-ICSI) In group II: 52 oocytes, conventional ICSI (C-ICSI) was performed as described previously (Van Steirteghem et al., 1993) In group II: 52 oocytes, conventional ICSI (C-ICSI) was performed as described previously (Van Steirteghem et al., 1993) Demirol et al, F&S, 2006

60. Material and methods After injection, oocytes incubated in IVF medium (Vitrolife) and covered with mineral oil After injection, oocytes incubated in IVF medium (Vitrolife) and covered with mineral oil Fertilization was controlled 16-18 h after the injection (Nagy et al., 1998) Fertilization was controlled 16-18 h after the injection (Nagy et al., 1998) Fertilized oocytes were transferred to G-1 medium(Vitrolife) and on day 2 they were transferred to G-2 medium (Vitrolife) Fertilized oocytes were transferred to G-1 medium(Vitrolife) and on day 2 they were transferred to G-2 medium (Vitrolife) Demirol et al, F&S, 2006

61. Material and methods Embryos were classified on day 3 according to the number of blastomers and percentage of fragmentation Embryos were classified on day 3 according to the number of blastomers and percentage of fragmentation Demirol et al, F&S, 2006

63. Results in LA-ICSI group Fertilization rate Fertilization rate embryos with > 6 cells embryos with > 6 cells <10% fragmentation <10% fragmentation Embryos < 4 cells Embryos < 4 cells D3 Significantly higher D3 Significantly lower Demirol et al, F&S, 2006

64. LA-ICSI related studies Nagy et al, 2001 Nagy et al, 2001 Rienzi et al, 2001 Rienzi et al, 2001 Abdelmassih et al, 2002 Abdelmassih et al, 2002 Demirol et al, 2006 Demirol et al, 2006 With high degeneration rate, failed ICSI cycles Lower degeneration and higher embryo quality Resulted in

65. Take home massages-I The introduction of laser technology seems to be a helpful to simplify the various technique of gamete The introduction of laser technology seems to be a helpful to simplify the various technique of gamete The question of safety is always an important point when introducing a new techique The question of safety is always an important point when introducing a new techique

66. Take home massages-II Laser is currently being used in assisted hatching and embryo biopsy for PGD Laser is currently being used in assisted hatching and embryo biopsy for PGD Laser assisted ICSI widens the potential applications of laser devices Laser assisted ICSI widens the potential applications of laser devices