Laser Use in ART

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,1992Antinori,1996Mon
  toidis,2001, Hisieh 2002, Primi 2004)
• PGD Microdissection of ZP with a laser system
  simlifies subsequent polar body biopsy or removal
  of blastomers (Veiga,1997Boada,1998Montag,1998,
  Rienzi 2004, Nagy 2005)
• Defragmantation
• Necrotic blastomere removal
• Sperm immobilization (Tadir,1989, Ebner 2001)
• Laser Assisted ICSI( Nagy,2001Abdelmassih,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

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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

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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)

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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., CMJ1999,Hsieh et
  al.,FS2002)
• 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 (MantoudisHR,2001)

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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 Tyrodes
  solution

Hsieh et al., 2002, Fertil Steril 2002 78
179-182
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Hsieh et al., 2002, Fertil Steril 2002 78
179-182
10
Balaban et al. 2002, Hum Reprod 17 1239-1243
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(No Transcript)
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H. Sallam et al, 2003, meta analysis
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FS 2007
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FS 2007
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Different types of laser AH
Comparison of quarter, partial and total laser AH
in selected infertility patients
Mantoudis 2001, HR
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Mantoudis et al, 2001 Hum Reprod 16 2182-2186
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Mantoudis et al, 2001 Hum Reprod 16
2182-2186
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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
(plt0.001)
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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)

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FS 2007
21
FS 2007
22
Laser Assisted AHA
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Laser Assisted Zona Drilling for PGD

• Procedure makes polar body biopsy more accurate
  and effective (Montag et al.,FS1998)
• Sucessful outcomes demonstrate the efficacy and
  safety of the laser assisted embryo biopy to
  facilitate PGD (Han et al.,FS2003)
• 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.,HR2003)

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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
  PGDResults 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)

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Laser PGD
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Laser DFRG
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Laser Sperm Immobilizasyon
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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

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• 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.

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• 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.

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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) (n410)
• RIF (2) (n796)
• Frozen/thawed ET (n180)

Valojerdi et al, FS in press
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Results with advanced age and RIF

• 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)

NO DIFFERENCE
Valojerdi et al, FS in press
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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, FS in press
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Laser assisted ICSINEW ECHNIQUE
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Conventional ICSI

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

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ICSI

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

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ICSI

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

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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 E when strong aspiration and/or
  reposition of injection needle is required to
  break the membrane

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Difficult Breakage Pattern

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

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Laser Systems

• Contact type- ERYAG (Obruca,1997)
• Noncontact - HoYSGG UV Laser (Liow,1996)
• 1480 mm diode laser (Germond,1996Rink,1996Montag
  ,1998Blake,2001)

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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,2001Nagy,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

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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

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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

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EASY and DIFFICULT ICSI
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Laser Assisted ICSI
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Case reports of LA-ICSI

• 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

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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
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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
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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)

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Laser Assisted ICSI can be applied to

• Oocytes showing an increased elasticity of the
  oolemma (Rienzi,2001Abdelmassih,2002)
• In oocytes showing an inherent fragility of the
  membrane (Abdelmassih,2002Nagy.2002)
• To rescue oocytes after failed fertilization with
  conventional IVF ( Eroglu,2002)
• Oocytes showing normal membrane response
  (Nagy, 2004Moser,2004)

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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

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

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Objective

• 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

Demirol et al, FS 2006
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Material and methods

• Forty patients (forty ICSI cyles)
• Four or less M II oocytes were retrieved
• Two or more failed ICSI cycles
• Mean age 38.6 yrs
• Demirol et al, FS 2006

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Material and methods

• 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)
• Oocytes were retrieved by transvaginal USG guided
  36 hours after HCG administration
• Stored in incubator at 37 0C with 5 CO2 for 2-4
  hours

Demirol et al, FS 2006
56
To ensure the maximum safety

• 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 innermost layer of the zona pellucida(ZP)
  (0.5µm) was kept intact
• very short pulse duration (lt2 ms) was applied
• No visible sign of oolemma reaction was observed

Demirol et al, FS, 2006
57
LA-ICSI was performed in the following manner-I

• Oocytes and spermatozoa were placed into an
  injection dish as usual
• 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 oclock
  position (where the injection needle would
  penetrate into the oocyte)

Demirol et al, FS 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)
• A channel with small diameter (5-6 µm) was
  drilled with 3-5 low energy pulses, always using
  lt2 ms of pulse duration
• The injection pipette was introduced through this
  channel and ICSI was performed as usual

Demirol et al, FS, 2006
59
Material and methods

• 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)

Demirol et al, FS, 2006
60
Material and methods

• 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)
• Fertilized oocytes were transferred to G-1
  medium(Vitrolife) and on day 2 they were
  transferred to G-2 medium (Vitrolife)

Demirol et al, FS, 2006
61
Material and methods

• Embryos were classified on day 3 according to the
  number of blastomers and percentage of
  fragmentation

Demirol et al, FS, 2006
62
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Resultsin LA-ICSI group

• Fertilization rate
• embryos with gt 6 cells
• lt10 fragmentation
• Embryos lt 4 cells

Significantly higher
D3
Significantly lower
D3
Demirol et al, FS, 2006
64
LA-ICSI related studies

• Nagy et al, 2001
• Rienzi et al, 2001
• Abdelmassih et al, 2002
• Demirol et al, 2006

With high degeneration rate, failed ICSI cycles
Resulted in
Lower degeneration and higher embryo quality
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Take home massages-I

• 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

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Take home massages-II

• Laser is currently being used in assisted
  hatching and embryo biopsy for PGD
• Laser assisted ICSI widens the potential
  applications of laser devices

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Thank you for your attention