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Using a laser system for Assisted
Hatching, Vitrification and PGD/PGS
Ronny Janssens
Outline



Validation of PDG/PGS @ UZB
Use of laser systems in ART








2

PGD
(Defragmentation)
(Sperm immobi...
PGD/PGS @ UZB

2/93 3/96 5/98

6/98

11/98

6/99

Decompaction media

First PGD
Aspiration of blastomeres

Sequential medi...
Mechanical opening
Compaction/decompaction


Ca2+/Mg2+ -free medium for decompaction

De Vos: Original image
Laser Systems


1.48 µm non-contact diode laser (Rink, 1996)
Laser power and pulse duration


High vs low energy laser



400mW laser
 200µs (=80µJ)


7

400µs

80µJ in 10µl dropl...
Acid Tyrode vs laser
AT



Double tool holder
Complicated and time
consuming

Laser



Fast setup
Simple and fast
proc...
Comparison laser with Acid Tyrode




9

Comparison of the results of human embryo biopsy and outcome of
PGD after zona ...
Blastomere damage
Acid Tyrode
drilling

Laser drilling

Aspirated blastomeres

681

775

Damaged blastomeres

33

13

Dama...
Comparison laser with Acid Tyrode
Acid Tyrode
 Complex set-up

Laser
 Simple set-up

 Double tool holder
 3 injectors
...
Zona drilling techniques

initially

currently
Latest developments in lasers







13

Movable laser
Programable path
Pulses spaced in time (500 msec)
Hands free r...
PGS by FISH at cleavage stage is not effective in
improving live birth delivery rate per cycle

“This RCT provides no argu...
Brussels PGD team PGS update


Rationale
 >40 years: >50% aneuploidy rate


Pellestero et al., 2003

 20-30 years: 20%...
Conclusions



PGS still is an expiremental procedure
More research is ongoing
 ESHRE CGH pilot study: proof of princip...
Outline



Validation of PDG/PGS @ UZB
Use of laser systems in ART








17

PGD
(Defragmentation)
(Sperm immob...
Timing of biopsy (ESHRE PGD
guidelines, Harton et al. 2011)


PB
 1st PB: 36-42h post hCG
 1 and 2nd PB: 9-22h post ins...
Polar body biopsy











19

Screening, no info about paternal meiotic errors or postzygotic
abnormalities
L...
Cleavage stage biopsy


Advantage
 Technically more straightforward
 Ample time
 Good concordance rate



Disadvantag...
Trophectoderm biopsy


Advantage








Disadvantage





21

Double selection
Less invasive
Less embryos for ...
22

30-1-2014
Assisted Hatching (Cohen, 1990)
 Hatching

failure or failure to escape from the
ZP is one of the reasons held responsibl...
Laser Assisted Hatching techniques


Opening
 Total laser AH - Single shot
 Total laser AH – multiple shots



Thinnin...
Carney et al. 2012 ochran AH toevoegen
“Whilst assisted hatching (AH) appeared to
offer a significantly increased chance o...
A comparison of four different techniques of assisted hatching
Balaban et al. HR 2002

PZD

Acid
tyrode

Diode laser

Pron...
AH: the existing evidence – Carney 2012



Good prognosis patients and unselected patients do not
benefit from AH (n=8)
...
Laser Assisted Imotile Sperm Selection
(LAISS)


Immotile sperm selection






HOS test (Liu et al, 1997)
MTT
Pento...
LAISS

(a)
(b)
(c)
•a,b,c: vital
•d: non - vital

(d)
30

30-1-2014
Vitrier et al, 2012


31

Fertilization rates obtained after ICSI with
immotile LAISS-positive sperm (28.9%) and
with imm...
Artificial shrinkage prior to vitrification


32

Iwayama 2010: In vitro and in vivo viability of human blastocysts
colla...
Blastocyst collapse






33

Laser: 1 pulse on the junction between TE
cells
Needle
Osmotic shock: incubator in 0,2 ...
Artificial shrinkage
Laser
collaps

Pre collaps

Post warming
15 min

34

1 hour

24 hours

30-1-2014
Conclusions


Consider new technologies as an
expiremental procedure
 Do you really need it?
 Validate it





35

T...
Thank you
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Using a Laser System for Assisted Hatching, Vitrification and PGD/ PGS - Ronny Janssens, Quality Manager, Centre for Reproductive Medicine at UZ, Brussels

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Ronny Janssens the Quality Manager at the Centre for Reproductive Medicne at UZB talked about using a laser system for assisted hatching, vitrification and PGD/PGS at the RI Istanbul Conference and Workshop on the 23rd and 24th January 2014.
Throughout this presentation he covers:
- Validation of PGD/ PGS @UZB
- Use of laser systems in ART
- Mechanical opening
- Compaction/ decompaction
- Laser power and pulse duration
- Acid tyrode vs laser
- Zona drilling techniques
- PGS by FISH at cleavage stage is not effective in improving live birth delivery rate per cycle
- Timing of biopsy
- Polar body biopsy
- Cleavage stage biopsy
- Trophectoderm biopsy
- Assisted hatching
- Laser assisted immotile sperm selection
- Artificial shrinkage prior to vitrification
- Blastocyst collapse
- Artificial shrinkage

Using a Laser System for Assisted Hatching, Vitrification and PGD/ PGS - Ronny Janssens, Quality Manager, Centre for Reproductive Medicine at UZ, Brussels

  1. 1. Using a laser system for Assisted Hatching, Vitrification and PGD/PGS Ronny Janssens
  2. 2. Outline   Validation of PDG/PGS @ UZB Use of laser systems in ART        2 PGD (Defragmentation) (Sperm immobilization) AH LAISS (LA ICSI) Vitrification 30-1-2014
  3. 3. PGD/PGS @ UZB 2/93 3/96 5/98 6/98 11/98 6/99 Decompaction media First PGD Aspiration of blastomeres Sequential media Acid Tyrode Laser zona drilling
  4. 4. Mechanical opening
  5. 5. Compaction/decompaction  Ca2+/Mg2+ -free medium for decompaction De Vos: Original image
  6. 6. Laser Systems  1.48 µm non-contact diode laser (Rink, 1996)
  7. 7. Laser power and pulse duration  High vs low energy laser  400mW laser  200µs (=80µJ)  7 400µs 80µJ in 10µl droplet: +0,002°C 30-1-2014
  8. 8. Acid Tyrode vs laser AT   Double tool holder Complicated and time consuming Laser   Fast setup Simple and fast procedure
  9. 9. Comparison laser with Acid Tyrode   9 Comparison of the results of human embryo biopsy and outcome of PGD after zona drilling using acid Tyrode medium or a laser. Joris, H., De Vos, A., Janssens, R., Devroey, P., Liebaers, I., and Van Steirteghem, A. Hum. Reprod. 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 Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study. Geber et al. Reprod Biol Endocrinol. 2011 30-1-2014
  10. 10. Blastomere damage Acid Tyrode drilling Laser drilling Aspirated blastomeres 681 775 Damaged blastomeres 33 13 Damage during zona drilling 19 0 Damage during cell manipulation 14 13
  11. 11. Comparison laser with Acid Tyrode Acid Tyrode  Complex set-up Laser  Simple set-up  Double tool holder  3 injectors    Training/skills required Time consuming Low precision     Not reproducable  Difficult training/QC  Chemical damage  Standardised procedure  Easy QC   Cellysis  11 No investment Short learning curve Fast procedure Precise and accurate Thermal damage  None  Expensive equipment
  12. 12. Zona drilling techniques initially currently
  13. 13. Latest developments in lasers      13 Movable laser Programable path Pulses spaced in time (500 msec) Hands free repeated laser shots Faster procedure 30-1-2014
  14. 14. PGS by FISH at cleavage stage is not effective in improving live birth delivery rate per cycle “This RCT provides no arguments in favour of PGD-AS for improving clinical outcome per initiated cycle in patients with AMA when there are no restrictions in the number of embryos to be transferred”   14 Confirmed by Mastenbroeck et al., 2007 Example of introduction into clinical practice without evidence 30-1-2014
  15. 15. Brussels PGD team PGS update  Rationale  >40 years: >50% aneuploidy rate  Pellestero et al., 2003  20-30 years: 20% oocyte aneuploidy rate  Fragouli et al., 2006  2/3 human cleavage stage embryos aneuploid  15 Delhanty et al., 1997; Magli et al., 2000; Bielanska et al., 2005; Mantzouratou et al., 2007 30-1-2014
  16. 16. Conclusions   PGS still is an expiremental procedure More research is ongoing  ESHRE CGH pilot study: proof of principle  ESHRE Study into The Evaluation of oocyte Euploidy by Microarray (ESTEEM trial)  16 Multicentre prospective randomised study into PB CGH vs no CGH 30-1-2014
  17. 17. Outline   Validation of PDG/PGS @ UZB Use of laser systems in ART        17 PGD (Defragmentation) (Sperm immobilization) AH LAISS (LA ICSI) Vitrification 30-1-2014
  18. 18. Timing of biopsy (ESHRE PGD guidelines, Harton et al. 2011)  PB  1st PB: 36-42h post hCG  1 and 2nd PB: 9-22h post insemination  Sequential removal of PBs on D0 and D1  Cleavage stage  Morning of D3  Blastocyst  Zona breaching on D3 and TE biopsy on D5  Zona breaching and TE biopsy on D5 18 30-1-2014
  19. 19. Polar body biopsy           19 Screening, no info about paternal meiotic errors or postzygotic abnormalities Lower diagnostic accuracy rate Female meiotic errors are the predominant cause of pregnancy loss, abnormal pregnancy, miscarriage and affected live births Provides important prognostic information on the orgin of aneuploidies Info on likelihood of pregnancy using their own eggs and wether to consider egg donation Less invasive More time for analysis Labour intensive Time-consuming Not all oocytes develop into viable embryos 30-1-2014
  20. 20. Cleavage stage biopsy  Advantage  Technically more straightforward  Ample time  Good concordance rate  Disadvantages  Chromosome instability/mosaicism  No definite conclusion on the basis of 1 cell 20 30-1-2014
  21. 21. Trophectoderm biopsy  Advantage       Disadvantage     21 Double selection Less invasive Less embryos for biopsy Lower mosaicism More DNA Labour intensive (variable stages of blastocysts) Time limitation for diagnosis Need for vitrification Effect on children insufficiently demonstrated 30-1-2014
  22. 22. 22 30-1-2014
  23. 23. Assisted Hatching (Cohen, 1990)  Hatching failure or failure to escape from the ZP is one of the reasons held responsible for low implantation rates in ART  Underlying causes for failed hatching are Naturally thick zonae pellucidae Zona hardening due to prolonged culture Lysin production failure (culture induced or intrinsic)
  24. 24. Laser Assisted Hatching techniques  Opening  Total laser AH - Single shot  Total laser AH – multiple shots  Thinning  Partial laser AH  LAQZT 24 30-1-2014
  25. 25. Carney et al. 2012 ochran AH toevoegen “Whilst assisted hatching (AH) appeared to offer a significantly increased chance of achieving a clinical pregnancy, the extent to which it might do so only just reached statistical significance. The ’take home’ baby rate was still not proven to be increased by AH, and multiple pregnancy rates were significantly increased in the AH groups. Most trials still failed to report on live birth rates.” 25 30-1-2014
  26. 26. A comparison of four different techniques of assisted hatching Balaban et al. HR 2002 PZD Acid tyrode Diode laser Pronase zona thinning No. of ET cycles 239 191 219 145 188 Mean age 34.4 34.6 35.0 34.5 29.8 No. of oocytes retrieved 9,6 9,8 9,5 9,7 12,3 No. of ET 3,8 3,9 4,0 3,7 3,4 IR (%) 18,6 17,4 18,9 19,1 21,6 CPR(%) 49.3 46.0 48.4 46.8 48.4 Retrospective, selected patients Controls
  27. 27. AH: the existing evidence – Carney 2012   Good prognosis patients and unselected patients do not benefit from AH (n=8) Two studies show a significant benefit for repeated implantation failure patients  Blastocyst (chemical removal) Jelinkova, 2002  D3 Es (LAQZT) (low nrs) Petersen, 2005b  Frozen embryos benefit significantly from AH  One study, LAQZT Balaban, 2006  AH is not indicated for  Female age Lanzendorf, 1998  Endometriose Ciray, 2005  Selective hatching on poor Q embryos Cohen, 1992b 27 30-1-2014
  28. 28. Laser Assisted Imotile Sperm Selection (LAISS)  Immotile sperm selection      HOS test (Liu et al, 1997) MTT Pentoxifylline (Yovitch et al., 1988) Theophilline LAISS (Aktan TM, Montag M, Duman S, Gorkemli H, Rink K, Yurdakul T Use of a laser to detect viable but immotile spermatozoa. Andrologia 2004;36:366-369)  Birefringence-polarisation microscopy (Collodel et al. 2010) 28 30-1-2014
  29. 29. LAISS (a) (b) (c) •a,b,c: vital •d: non - vital (d)
  30. 30. 30 30-1-2014
  31. 31. Vitrier et al, 2012  31 Fertilization rates obtained after ICSI with immotile LAISS-positive sperm (28.9%) and with immotile MTT-selected sperm (31.9%) were not significantly different. 30-1-2014
  32. 32. Artificial shrinkage prior to vitrification  32 Iwayama 2010: In vitro and in vivo viability of human blastocysts collapsed by laser pulse or osmotic shock prior to vitrification 30-1-2014
  33. 33. Blastocyst collapse      33 Laser: 1 pulse on the junction between TE cells Needle Osmotic shock: incubator in 0,2 M sucrose solution for 1-2 min Physical manipulation Biopsy 30-1-2014
  34. 34. Artificial shrinkage Laser collaps Pre collaps Post warming 15 min 34 1 hour 24 hours 30-1-2014
  35. 35. Conclusions  Consider new technologies as an expiremental procedure  Do you really need it?  Validate it    35 The use of lasers for Zona opening is easier and safer than AT Lasers may be usefull for AH and artificial shrinkage prior to vitrification More research needed 30-1-2014
  36. 36. Thank you

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