Are Laser Zona Drilling and
Embryo Biopsy
safe procedures?
Katerina Chatzimeletiou B.Sc, M.Sc, Ph.D
Assistant Professor in...
PB Biopsy
18h

D4

D2

Cleavage Biopsy
D3

D2

D5

Blastocyst Biopsy

D7
Is Acid Tyrode’s drilling safe?
PI

CFSE

TE
ICM

A

B

Chatzimeletiou K, Picton H, Handyside A.H 2001 Reproductive Biomed...
Is laser zona drilling with infrared laser safe?
Differential Labelling of LASER Drilled Mouse Embryos
__________________________________________________

Control
(Day 6)
...
Immediate effects of Laser Zona Drilling in the mouse

f

g

m

l

h

i

o

n

j

k

q

p

Chatzimeletiou K, Picton H, Han...
Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
Viability staining after zona drilling in human preimplantation embryos
__________________________________________________...
Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
Chatzimeletiou et al., 2014 Unpublished data

Damage is not caused when the laser is fired
at a distance ≥ 5um at 0.4ms
Embryo Biopsy (Day 3 - cleavage stage)

Κ. Χατζημελετίου - Bιογένεσις
Preimplantation Genetic Screening (PGD/S)

Κ. Χατζημελετίου - Bιογένεσις

Genetic Analysis
By FISH

Genetic Analysis
by PC...
Τοποθέτηση του Fluorescent Probe

Πλύσεις

DNA Denaturation στους 75oC/ 5 min &
υβριδισμός στους 37oC/ 3hours
Preimplantation Genetic Screening (PGS) for Aneuploidy
21

18

22
18

16
16

22
13

21
13

Normal Diploid
X
Y
15

15

Norm...
PGS does not increase pregnancy rates
Critique - Mastenbroek et al (2007) NEJM
•
•

ET involving undiagnosed embryos (20%)
Some 4 cell embryos biopsied

•
•

FI...
Preimplantation Genetic Screening (PGS) for Aneuploidy
No Implantation

Normal Diploid

Impaired Endometrial Receptivity?
...
Array CGH

_________________________________________________________
Amplified
DNA
reference
Sample

Scanner

Software
BlueGnome 24sure V3 – Array CGH
in 2014

Time taken
30 min

Whole Genome Amplification

2 hr. 30 min

Labelling

1 hr.

Co...
PGDIS
Array – CGH (day 3) Biopsies
PGS Clinical data
No. of cycles
No. of couples
Maternal Age (average)
No. of embryos an...
Results

Day 3 Biopsy

Day 5 Biopsy

Implantation

Age

(+ Sac)

Implantation

Aneuploidy Rate

Age

(+ Sac)

Aneuploidy R...
Blastocysts biopsy and aCGH eliminates
the maternal age effect on implantation
Day 3 biopsy*

100%
90%
80%
70%
60%
50%
40%...
Blastocysts biopsy and aCGH eliminates
the maternal age effect on implantation
Day 5 biopsy

90
80
60

implantation rate
p...
CLINICAL TRIALS
 First published 24sure RCT (Yang et al 2012)
 65% increase in 20 week pregnancy rate in 24sure treatmen...
CLINICAL TRIALS
Spindle abnormalities in human embryos
Tetrapolar Spindle

Congression Failure

Chatzimeletiou et al., 2005 Hum. Reprod. 2...
Tripolar spindles in human blastocysts

Chatzimeletiou et al., 2005 Hum Reprod 20 (3):672-682
Tripolar and Normal spindles in the TE of a day 5 human blastocyst

Chatzimeletiou et al., 2005 Hum. Reprod. 20 (3):672-68...
PGS in 2014
• Aim for safer biopsy
• Transfer of normal embryos and increase in
implantation and pregnancy rates
• Reduce ...
Cleavage biopsy (day 3)

Blastocyst

Chatzimeletiou et al., 2012 PGDIS Austria

Vitrification
Hum Reprod (2012) 27 (1): 106 – 113.

51/55 (92.7%) vitrified blastocysts
survived

120/175 (68.6%) spindles had normal
sh...
Confocal section of a human vitrified blastocyst showing an
anaphase spindle with chromosome bridging

Chatzimeletiou et a...
Conclusions
Laser zona drilling and embryo biopsy are safe procedures as long
as safe working distances are kept.

PGS has...
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Are Laser Zona Drilling and Embryo Biopsy Safe Procedures?

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Are Laser Zona Drilling and Embryo Biopsy Safe Procedures?

  1. 1. Are Laser Zona Drilling and Embryo Biopsy safe procedures? Katerina Chatzimeletiou B.Sc, M.Sc, Ph.D Assistant Professor in Embryology and Genetics IVF Unit, 1st Department of Obstetrics & Gynaecology, Aristotle University Medical School, Papageorgiou General Hospital, Thessaloniki, Greece
  2. 2. PB Biopsy 18h D4 D2 Cleavage Biopsy D3 D2 D5 Blastocyst Biopsy D7
  3. 3. Is Acid Tyrode’s drilling safe? PI CFSE TE ICM A B Chatzimeletiou K, Picton H, Handyside A.H 2001 Reproductive Biomedicine Online,2 (3): 178- 187
  4. 4. Is laser zona drilling with infrared laser safe?
  5. 5. Differential Labelling of LASER Drilled Mouse Embryos __________________________________________________ Control (Day 6) Laser (Day 6) Laser (Day 5) Laser (Day 5) Chatzimeletiou et al., 2001 Reproductive Biomedicine Online,2 (3): 178- 187
  6. 6. Immediate effects of Laser Zona Drilling in the mouse f g m l h i o n j k q p Chatzimeletiou K, Picton H, Handyside A.H 2001 Reproductive Biomedicine Online,2 (3): 178- 187
  7. 7. Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
  8. 8. Viability staining after zona drilling in human preimplantation embryos __________________________________________________________ Acid Tyrode’s PI CFSE Infrared Laser CFSE Chatzimeletiou et al., 2005 RBM Online 11(6):697-710 PI
  9. 9. Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
  10. 10. Chatzimeletiou et al., 2005 RBM Online 11(6):697-710
  11. 11. Chatzimeletiou et al., 2014 Unpublished data Damage is not caused when the laser is fired at a distance ≥ 5um at 0.4ms
  12. 12. Embryo Biopsy (Day 3 - cleavage stage) Κ. Χατζημελετίου - Bιογένεσις
  13. 13. Preimplantation Genetic Screening (PGD/S) Κ. Χατζημελετίου - Bιογένεσις Genetic Analysis By FISH Genetic Analysis by PCR or Array- CGH
  14. 14. Τοποθέτηση του Fluorescent Probe Πλύσεις DNA Denaturation στους 75oC/ 5 min & υβριδισμός στους 37oC/ 3hours
  15. 15. Preimplantation Genetic Screening (PGS) for Aneuploidy 21 18 22 18 16 16 22 13 21 13 Normal Diploid X Y 15 15 Normal Male
  16. 16. PGS does not increase pregnancy rates
  17. 17. Critique - Mastenbroek et al (2007) NEJM • • ET involving undiagnosed embryos (20%) Some 4 cell embryos biopsied • • FISH quality? No data on error rates provided Chromosomes 15 and 22 not included (account for 10% abnormalities) • Cumulative data from 3 cycles (not good clinical management) • AMA (35-41 yrs) heterogeneous group
  18. 18. Preimplantation Genetic Screening (PGS) for Aneuploidy No Implantation Normal Diploid Impaired Endometrial Receptivity? Abnormalities in other chromosomes? Embryonic mosaicism? Normal Male
  19. 19. Array CGH _________________________________________________________ Amplified DNA reference Sample Scanner Software
  20. 20. BlueGnome 24sure V3 – Array CGH in 2014 Time taken 30 min Whole Genome Amplification 2 hr. 30 min Labelling 1 hr. Combination and volume reduction 3 hr. 30 min •Aneuploidy screening to improve IVF success rates & reduce miscarriage rates Preparation 2 hr. PGS Protocol Steps Hybridization 30 min Washing 30 min Scanning 1 hr. Analyse and report
  21. 21. PGDIS Array – CGH (day 3) Biopsies PGS Clinical data No. of cycles No. of couples Maternal Age (average) No. of embryos analysed Mean no. of embryos analysed per cycle No. of embryos diagnosed ANEUPLOID NORMAL No. of embryos with diagnosis failure No. of embryos transferred Mean no. of embryos transferred No. of transfers No. +HCG (% per ET ) No. of biochemical pregnancies No. of miscarriages No. of Clinical pregnancies (% per ET ) Age (average) No. of Fetal Sacs No. of Fetal Hearth Beat (FHB) Implantation rate Total No. (%) 170 155 38.4±3.9 1264 7.4±3.5 1171 (92.6) 882 (75.3) 289 (24.7) 93 (7.4) 179 1.5±0.6 115 (67.6) 80 (69.6) 3 2 75 (65.2) 37.2±3.2 91 88 50.8%
  22. 22. Results Day 3 Biopsy Day 5 Biopsy Implantation Age (+ Sac) Implantation Aneuploidy Rate Age (+ Sac) Aneuploidy Rate 30-34 51.7 (122/236) 48.5 (225/464) 30-34 63.2 (36/57) 34.2 (55/161) 35-39 40.1 (131/327) 64.5 (855/1325) 35-39 60.6 (57/94) 45.0 (175/389) 40-42 32.4 (70/216) 79.0 (845/1070) 40-42 61.5 (32/52) 65.0 (154/237) p<0.001 p<0.001 N.S. p<0.001
  23. 23. Blastocysts biopsy and aCGH eliminates the maternal age effect on implantation Day 3 biopsy* 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% implantation rate aneuploidy rate cycles with all abnormal <35 35-39 40-42 43-45 Day 5 data: CGH and aCGH data, Day 3 data: from aCGH, *cycles with >4 embryos biopsied
  24. 24. Blastocysts biopsy and aCGH eliminates the maternal age effect on implantation Day 5 biopsy 90 80 60 implantation rate per replaced embryo 50 aneuploidy rate 70 40 30 cycles with all embryos abnormal 20 10 0 30-34 35-38 39-42 43-45 Day 5 data: CGH and aCGH data, Day 3 data: from aCGH, *cycles with >4 embryos biopsied
  25. 25. CLINICAL TRIALS  First published 24sure RCT (Yang et al 2012)  65% increase in 20 week pregnancy rate in 24sure treatment arm  Fresh Single Embryo Transfer on day 6 in good prognosis patients
  26. 26. CLINICAL TRIALS
  27. 27. Spindle abnormalities in human embryos Tetrapolar Spindle Congression Failure Chatzimeletiou et al., 2005 Hum. Reprod. 20 (3):672-682
  28. 28. Tripolar spindles in human blastocysts Chatzimeletiou et al., 2005 Hum Reprod 20 (3):672-682
  29. 29. Tripolar and Normal spindles in the TE of a day 5 human blastocyst Chatzimeletiou et al., 2005 Hum. Reprod. 20 (3):672-682
  30. 30. PGS in 2014 • Aim for safer biopsy • Transfer of normal embryos and increase in implantation and pregnancy rates • Reduce time to achieve a pregnancy • The first results following array Analysis are encourraging!! AT WHICH STAGE IS BIOPSY SAFER?
  31. 31. Cleavage biopsy (day 3) Blastocyst Chatzimeletiou et al., 2012 PGDIS Austria Vitrification
  32. 32. Hum Reprod (2012) 27 (1): 106 – 113. 51/55 (92.7%) vitrified blastocysts survived 120/175 (68.6%) spindles had normal shape and chromosome alignment.
  33. 33. Confocal section of a human vitrified blastocyst showing an anaphase spindle with chromosome bridging Chatzimeletiou et al 2012 Human Reproduction 27 (1): 106 – 113.
  34. 34. Conclusions Laser zona drilling and embryo biopsy are safe procedures as long as safe working distances are kept. PGS has a double role Diagnostic  PGS + Follow-up Analysis Therapeutic ? We need more RCTs.......... The use of more advanced techiques (microarrays) and next generation sequencing may increase pregnancy rates

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