Transcript The Assisted Conception Unit The Assisted Conception Unit Optimising IVF success Pre-treatment work up Embryo selection Implantation “Older age” group Repetitive IVF failure.

The Assisted Conception Unit
The Assisted Conception Unit
Optimising IVF success
Pre-treatment work up
Embryo selection
Implantation
“Older age” group
Repetitive IVF failure
The Assisted Conception Unit
Static Tests
Endocrine
Basal FSH
Toner JP et al Fertil Steril 1991
Inhibin – B
Seifer DB et al Fertil Steril 1997
Anti Mullerian Hormone
Van Roiij IA et al Hum Reprod 2002
Biophysical
Antral follicle count
Chang MY et al Fertil Steril 1998
The Assisted Conception Unit
Assessment of ovarian reserve
Dynamic tests :
Gn - RHa stimulation test ( Δ E2 )
Ideal treshold value with ROC was < 9.5 iu for FSH,
> 180 pmol/L for Δ E2
Ranieri DM et al ; Fertil Steril 1998
The Assisted Conception Unit
Gonadotrophin Ovarian Stimulation test ( GOST)
75 IVF patients
FSH
Δ E2
Δ Inhibin B
AMH
AFC
Day 3 FSH vs number of eggs collected
FSH (mIU/ml)
30
r=0.2
NS
20
10
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules of FSH
Delta E2 (day 4-day 3) vs number of eggs collected
r=0.35
P=0.003
1000
Delta E2
800
600
400
200
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules
of FSH
Antral Follicles
Antral follicle count vs number of eggs collected
Antral follicle number
30
r=0.67
P<0.001
20
10
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules of FSH
Day 3 AMH vs number of eggs collected
70
r=0.5
P<0.001
60
AMH (pmol/l)
50
40
30
20
10
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules of FSH
Day 3 inhibin B vs number of eggs collected
r=0.41
P<0.001
Inhibin B (pg/ml)
500
400
300
200
100
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules of FSH
Delta inhibin B (day 4-day 3) vs number of eggs collected
800
r=0.6
P<0.001
Delta inhibin B
600
400
200
0
0
200
400
600
800
1000
Eggs collected x1000/number of ampoules of FSH
Summary
Antral follicle count is the best single biophysical marker of ovarian
reserve (r=0.67, P<0.001)
Problem: subjective
Muttukrishna et al, BJOG 2005
Summary
AMH is the best basal biochemical marker of ovarian reserve
R=0.5, P<0.001
Muttukrishna et al, BJOG 2005
Anti Mullerian Hormone
(AMH)
•
Optimal Fertility:
28.6 pmol/L - 48.5 pmol/L
•
Satisfactory Fertility:
15.7 pmol/L - 28.6 pmol/L
•
Low Fertility:
2.2 pmol/L - 15.7 pmol/L
•
Very Low/Undetectable
0.0 pmol/L - 2.2 pmol/L
Efficacy of Combined Markers
Markers vs eggs collected/amp
FSH
Regressio
n coefficient
Significance
Day 3 FSH + delta E2
0.37
P<0.01
Day 3 FSH + delta E2 + AFC
0.7
P<0.001
Day 3 FSH + Delta E2 + AFC +
delta inhibin B
0.8
P<0.001
Day 3 FSH + delta E2 + AFC +
AMH
0.73
P<0.001
AFC + delta inhibin B
0.78
P<0.001
AFC + day 3 AMH
0.7
P<0.001
AFC+ delta inhibin B+day 3 AMH
0.78
P<0.001
The Assisted Conception Unit
Assessment of ovarian reserve
Cumulative scoring system using : Age, FSH ,AMH AFC, ΔE2 and Δ
Inhibin → 87% sensitivity
80% specificity
Muttukrishna S et al Br.J.Obstet.Gynaecol 2005
Number of Antral Follicle count
Clinical Pregnancy rate %
1-4 (n=11)
18.2 %
5-9 (n=31)
32 %
≥ 10 ( 27)
48 %
The Assisted Conception Unit
Optimising ovarian stimulation
Treatment strategies for “poor responders ”
and patients with raised baseline FSH
Poor responders
Age and non-age related poor ovarian reserve
Short follicular phase: Limited follicular recruitment
Increased sensitivity to suppressive effects of CL
Surrey ES et al Fertil Steril 2000
Speroff L et al 1999
McNatty KP et al J Clin Endocrinol Metab 1979
Biljan m et al Fertil Steril 1998
GnRH agonists
Mode of action:
• Initial flare up effect
• Loss of receptors → native GnRH receptors excluded
from receptor binding
( desensitization)
The Assisted Conception Unit
Poor responders
GnRH receptors are present in ovarian granulosa cells
Liscoviyich M . J. Biol.Chem. 1999
Long down -regulation with GnRH-a :
* High dose of gonadotrophins
* Lower no of eggs retrieved
Hazout A et al Fertil Steril , 1993
Poor responders
Microdose GnRH-a flare-up regime
Scott RT at al ;Fertil Steril 1994
GnRH - agonists in the luteal phase only
Ranieri DM et al ;Hum Reprod 2001
GnRH antagonists
Mode of action:
• Competitive binding with GnRH at receptor site
• Rapid decrease in FSH, LH
• No initial flare up
Poor responders
GnRH antagonist :
Follicular phase
Craft I et al ;Hum Reprod 2001
Mid Luteal
Humaidan P et al , Reprod Biolmed Online 2005
Poor responders
Prospective randomised study
Comparison of microdose 40 mcg / day GnRH-a flare
up regime and GnRh antagonist ( 48 patients ) :
similar clinical outcome .
Akman M et al Hum Reprod 2001
Poor responders
Prospective randomised study
62 expected poor responders
Ovarian stimulation with 150 or 300 iu FSH
• Similar median number of oocytes
• No difference in pregnancy rates
Klinkert E et al Hum Reprod 2005
Morphological predictors of implantation
Oocyte morphology
Serhal P et al ; Hum Reprod 1998
PB abnormalities
Oritz et al; Gamete Res 1983
PN nucleoli morphology
Tesarik J et al; Hum Reprod 1999
PN morphology
Gianaroli L et al; Fertil Steril 2003
Early cleavage check
Neuber E et al; Hum Reprod 2003
Multinucleation
Balakier H et al Hum Reprod 1997
The Assisted Conception Unit
Oocyte cytoplasmic abnormalities : vacuoles ,
inclusion bodies.
Normal fertilisation
Normal cleavage
Normal embryo morphology
Poor pregnancy rate
Serhal P et al; Hum Reprod 1998
The Assisted Conception Unit
Predictors of implantation
PN nucleoli morphology
Tesarik J et al; Hum Reprod 1999
The Assisted Conception Unit
The Assisted Conception Unit
The Assisted Conception Unit
Morphological predictors of implantation
PN morphology
Gianaroli L et al; Fertil Steril 2003
Pronuclear morphology and chromosome complement
A. Centralised
Juxtaposed
32% normal
B. Non-centralised
Juxtaposed
41% normal
C. Centralised
Non-juxtaposed
0% normal
D. Different Size
0% normal
Gianaroli L et al; Fertil Steril 2003
E. Fragmented
0% normal
The Assisted Conception Unit
The Assisted Conception Unit
Polar body alignment and chromosome complement
. Longitudinal
36% normal
. Perpendicular
33% normal
. Others
7% normal
Gianaroli et al.,Fertil Steril 2003
Fragmentation
Highly fragmented embryos( > 35%) : significant reduction in
implantation rate.
Ebner T et al Hum Reprod update 2003
Staessen C et al Fertil Steril 1992
Alikani M et al Fertl Steril 1999
Early cleavage
Early cleavage : An indictor of subsequent
good quality blastocyst formation.
Neuber E et al, Hum Reprod 2003
Milki A et al , Fertil Steril 2002
Multinucleation
Multinucleation on Day 2 ( even with one affected
blastomere)
Significant reduction in blastocyst formation,
blastocyst quality, pregnancy and implantation rate
Very poor live birth rate.
Alikani M et al , Hum Reprod 2000
Balakier H et al Hum Reprod 1997
Uneven cell size
Uneven blastomeres have a negative influence
on pregnancy rates and implantation.
Morgan K et al , Hum Reprod 1995
Ziebe S et al , Hum Reprod 1997
Scott L et al. Hum Reprod 2007
Scott L et al Fertil Steril 2007
The Assisted Conception Unit
Cumulative scoring system is highly predictable
of pregnancy outcome
Oocyte morphology
NPB scoring
PN scoring
Early cleavage check ( 24 - 27 hrs )
Multinucleation
Cell size
The Assisted Conception Unit
A new system “ EmbryoGuard” for time laps
evaluation within the incubator.
High Magnification Selection Of Individual
Sperm
Vacuolated
Normal
• Pregnancy rate :
18%
45%
• Miscarriage rate:
100%
10%
• Live birth rate:
0%
41%
P ≤ 0.01
Bartov B et al ESHRE 2008
The Injection Of Acrosome Reacted Sperm In
Severe OAT
Non Reacted
Reacted
Pregnancy rate
11%
52%
P=0.002
Implantation Rate
8.6%
35%
P=0.002
Magli M C et al ESHRE 2008
The Assisted Conception Unit
Biomarkers of implantation in the supernatant of
individually cultured embryos
NIR spectroscopy:
Metabolomic profiling of oocytes
Unique metabolomic profiles of the specific OM
biomarkers populations identified.
MI and MII oocytes profiles were significantly
different from each other and from GV oocytes.
Specificity 91% ( MI) and 100% ( MII)
Nagy ZP et al ASRM 2007
NIR spectroscopy:
Metabolomic profiling of Blastocyst (SET)
Biomarkers of oxidative metabolism ( ROH,C=C,
-SH and – NH)
Statistically significant difference in changes of
biomarkers of OM between embryos that resulted
in clinical pregnancies vs embryos that failed to
implant.
Tucker M et al ASRM 2007
The Assisted Conception Unit
Predictors of implantation
Embryo competency
A combined morphometric and metabolomic assessment of the
developing embryo : major paradigm shift in ART
Optimising IVF success
To blastocyst or not to blastocyst ?
Blastocyst Transfer
ADVANTAGES
•
Synchronisation with the female tract (Olivennes et al., 1994; Ertzeid et al. 1993)
• Observation of embryonic genome activation ( Braude P et al 1988)
- Embryonic genome is activated at the 4- 8 cell stage
• De-selection of chromosomally abnormal embryos (Rubio et al., 2007)
• Postulated reduction of embryo expulsion (Lesney et al. 1998)
- Reduced uterine junctional zone contractions
•
Diagnostic
•
SET
•
Key Performance Indicator
Blastocyst Transfer
DISADVANTAGES
• Increased cycle cancellation
(Marek et al., 1999)
• Increased monozygotic twinning (Jain et al., 2004)
• Increased laboratory workload
– Embryo monitoring
– Weekend transfers
• Increased cost
– Culture medium
The Assisted Conception Unit
Day 2 vs Day 5 transfer
Prospective randomised study
CPR /EC was comparable : 32 % day 2 versus 44 %
day 5
60 % CPR/ ET on day 5 versus 35 % on day 2 P <
0.01
Van Der Auwera I et al Hum Reprod 2002
Prospective randomised studies
Day 3 vs Day 5
Significantly higher pregnancy rate with Day
5 transfer
 E.G. Papanikolaou et al. Fertil Steril ( 2005)
 Eliahu Levitas et al.Fertil Steril (2004)
 Raja Z. et al. Fertil Steril (2002)
 Gardner D K et al Fertil Steril 1998
Prospective randomised Studies
Day 3 vs Day 5
Embryo transfer is equally effective at cleavage stage and
blastocyst stage.
Hreinssona J, et al. (2004) European J Obst Gynecol
Coskun S et al Hum Reprof 2000
Scholtes etal Fertil Steril 1996
|
UCH DAY 3 Criteria for Blastocyst Transfer
– 4 x7/8 cell (<10% fragmentation) + at least one 6 cell embryo
(<20% fragmentation)
– 4 x 7/8 cell (<10% fragmentation). Good compaction in all
embryos
– 6 x 6 cells or more (< 20% fragmentation)
– 8 x 6 cells or more (>20< 50% fragmentation)
• Decision making evolves with confidence in the culture
system
2007 Fresh Embryo Transfers; Experience at UCH
Day Fresh
of No.IVF
ETs transfers
PR
ET
Day 3
Day 5
Day 6
58
83
21
CPR
Fresh ICSI transfers
IR
36/58
(62.1%)
31/58
(53.4%)
37/119
(31.1%)
68/83
(81.9%)
62/83
(74.7%)
87/165
(52.7%)
11/21
(52.4%)
11/21
(52.4%)
15/40
Day of
ET
No. ETs
PR
CPR
IR
Day 3
74
31/74
(41.9%)
25/74
(33.8%)
32/149
(21.5%)
Day 5
33
27/33
(81.8%)
25/33
(75.8%)
40/66
(60.6%)
Day 6
16
11/16
(68.8%)
10/16
(62.5%)
16/30
(53.3%)
(40.5%)
Av. eggs 11.7; Fertilisation 69.8%; Av. Age 35.5
Av eggs 9.4; Fertilisation 59.3%; Av Age 35.0
Multiple Pregnancy ; Experience at UCH
IVF
ICSI
Day of ET
Singleton
Twin
Triplet
MPR per ET
3
25
6
0
6/31 (19.4%)
5
36
24
1 MZ
25/61 (41.0%)
6
8
2
1 MZ
3/11 (27.3%)
Day of ET
Singleton
Twin
Triplet
MPR
3
18
7
0
7/25 (28.0%)
5
11
13
1 MZ
14/25 (56.0%)
6
4
6
0
6/10 (60.0%)
The Assisted Conception Unit
Improving implantation
Salpingectomy (hydrosalpinges)
The problematic cervix
Intrauterine pathology
Mid to low cavity ET
Surgical treatment of tubal disease prior to IVF
Three randomised clinical trials ( 295 patients)
The odds of live birth ( OR 2.13, 95% confidence interval
1.24 to 3.65) were increased with laparoscopic salpingectomy for
hydrosalpinges prior to IVF
Cochrane Database Syst Rev.2004
Uterine Polyposis
Can endometrial polyps affect fertility?
Retrospective study: small group of patients
pregnancy rate (78%)
Varasteh NN et al ;Obstet Gynaecol 1999
80 % pregnancy rate after polypectomy
Speiwankiewicz B et al;Clin Exp Obtet Gyaecol 2003
NO RCT
The Assisted Conception Unit
TVS vs HYCOSY
TVS could not :
Confidently diagnose submucous fibroids in the presence of a
uterus with multiple fibroids
Distinguish between hyperplastic endometrium and a large polyp
Differentiate between an arcuate uterus and a septate uterus
Ayida G et al ; Ultrasound Obstet Gyncol 1997
The Assisted Conception Unit
HYCOSY
86 consecutive patients : regular cycles ,
normal baseline scan
17 patients ( 20 % ) : Hycosy abnormal
9 patients ( 10.5 %) : Intra-uterine pathology detected
Factors affecting ET technique and its outcome
Experience and dexterity of the clinician
Hearns Stockes RM et al Fertl Steril 2000
Catheter type.
Schoolcraft WL et al Fetil Steril 2001
Placement of catheter tip in mid – low fundal area
Coroleu et al, Hum Reprod 2002
Olivera et al 2004 RBM on line 2004
Presence of cervical stenosis
Mansour R et al Fertil Steril 1990
Technically difficult ET → significant reduction
in pregnancy rates
Leeton J et al, Fertil Steril 1982
Wood C et al , Fertil Steril 1985
Englert Y et al, J Ass In Vitro Fertil Embryo Transfer 1986
Diedrich K et al, Hum Reprod 1989
Visser DS et al, J Reprod Genet 1993
Goudas V etal, Fertil Steril 1998
The Assisted Conception Unit
The problematic cervix
Full bladder to straighten out the uterine cavity .
The use of tenaculum to straighten the utero-cervical angle .
Use of a catheter with a rigid stylet.
Ultrasound guided transfer technique to visualise the position
of the catheter .
Transmyometrial ET .
Laparoscopic tubal ET .
Cervical dilatation .
The Assisted Conception Unit
The problematic cervix
Cervical Dilatation
Cervical dilatation two days before ET
No pregnancies were achieved .
Visser DS et al 1993
Cervical dilatation during ovum pick-up :
Poor pregnancy rate ( 2.5%)
Groutz A et al Fertil Steril 1997
The Assisted Conception Unit
The problematic cervix
Cervical dilatation under GA before starting gonadotrophin
stimulation : 59 treatment cycles ; easier ET and improved
pregnancy rate 32% .
Abusheikha N et al Fertil Steril 1999
Dilapan –S Osmotic Cervical Dilators ( hydrogel rod ): Diameter increases
from 3 to 8-10 mm within 4 hrs .
Serhal P et al ; Hum Reprod Dec 2003
The Assisted Conception Unit
Dilapan –S Osmotic Cervical Dilators
54 cycles : Dilapan –S inserted on the day of starting gonadotrophin
stimulation ; easier ET and 55 % clinical pregnancy rate .
Serhal P et al ; Hum Reprod Dec 2003
The Assisted Conception Unit
Cervical dilatation under GA vs Dilapan insertion
Bourne Hall study ( 57 patients ) :
40 ( 70.2 % ) subsequent ET was easy
17 ( 29.8 % ) ET remained difficult
UCLH study ( 54 patients ) :
43 ( 79 .6 % ) subsequent ET was easy
11 ( 20. 4% ) ET remained difficult
The Target
Pope et al
Fertil Steril 2004
©
Oliviera et al RBM on line
2004
Frankfurteret al Fertil Steril
2004
Coroleu et al Hum Reprod
2002
Repetitive failure IVF
No change in the pregnancy rate over the first 3
IVF cycles.
Pregnancy rate decreases by 40% thereafter
Templeton A N Engl Med 1998
Repetitive failure IVF
•
•
•
•
•
•
Blastocyst transfer
Tubal transfer( ZIFT/GIFT)
Assisted hatching
Co – culture
Media supplemented with growth factors
???Aneuploidy screen
Repetitive IVF failure
Blastocyst vs ZIFT
Prospective study
Mean no of 6.9 ± 3.7 failed attempts
64 patients ( 32 in each arm)
Age:
IVF (33.4 ± 5.2)
ZIFT( 33.5 ± 5.9 )
LBR per cycle : 0 % ( blastocyst ) 38.7 % ( ZIFT)
P.0004
Multiple pregnancy rate 54% (ZIFT)
Levran D et al ,Fertil Steril 2002
Repetitive IVF failure
Co – Culture improve outcome in patients with
history of poor embryo development.
Spandorfer SD et al J Assist Genet 2002
Repetitive IVF failure
Media supplemented with growth factors or cytokines:
Faster developing embryos
Embryos with more cells
Embryos with decreased apoptosis
Behr B, isivf 2007
Recurrent IVF Failure
Aneupoidy screen
Improved outcome in younger women
High cancellation rate and low cycle outcome
in women ≥ 40 years of age
Gianaroli L et al ;Fertil Steril 1999
Kahraman S et al, Human Reprod 2000
Munne S et al, Reproductive Biomed Online 2003
Caglar GS et al, Reproductive Biomed Online 2005
Preimplantation Genetic Screening
(PGS)
Embryo Biopsy
FISH probes
15
13
12
13
16
13
12
11. 2
11. 1
11
12. 1
12. 2
12. 3
13
14. 1
14. 2
14. 3
21. 1
21. 2
21. 3
22
31
32
33
34
13. 3
13. 2
13. 1
21
12
11. 2
11. 1
11. 1
11. 2
12. 1
12. 2
13
21
22
23
24
13
12
11. 2
11. 1
11. 1
11. 2
21
22. 1
22. 2
22. 3
11. 2
11. 1
11. 1
11. 2
12
13
14
15
21. 1
21. 2
21. 3
22. 1
22. 2
22. 3
13
18
11. 32
11. 31
11. 1
11. 1
11. 2
12. 1
12. 2
12. 3
21. 1
21. 2
21. 3
24
25
26. 1
26. 2
26. 3
22
11. 2
22
23
13
12
11. 2
11. 1
11. 1
11. 2
12. 1
12. 2
12. 3
13. 1
13. 2
13. 2
FISH
Green – chromosome 13
Red – chromosome 21
Normal
Four copies
One 21
Three 13
The Assisted Conception Unit
Assisted hatching
Systematic review : 23 randomised clinical trials (6 reported LBR)
No significant effect on LBR ( OR 1.21, 95% CI 0.82-1.78 )
Significant effect on clinical pregnancy in women after failed cycle(s)( OR 2.33 )
Only two studies included women > 37 . Meta- regression : AH might be useful
in older women ( although falling just short of statistical significance )
Edi-Osagie E et al ; Hum Reprod 2003
Repetitive IVF failure
Smoking:
Direct impact on IVF success rate
Reduction in ovarian reserve (↓AMH levels)
Barriere P et al ASRM 2007
The Assisted Conception Unit
Optimising IVF success in the older age group
CAN SCIENCE BEAT THE BIOLOGICAL CLOCK ?
The Assisted Conception Unit
Biological ageing of the egg is a physiological process
Oocyte aneuploidy
Embryonic aneuploidy
Mitochondrial DNA point mutations
The Assisted Conception Unit
Biological ageing of the egg is a physiological process
Oocyte aneuploidy
Embryonic aneuploidy
Mitochondrial DNA point mutations
The Assisted Conception Unit
OOCYTE ANEUPLOIDY
79 % of oocytes of older women exhibited abnormal meiotic spindle
(abnormal tubulin placement and one or more chromosomes were
displaced from the metaphase plate during the second meiotic
division )
Only 17 % of oocytes in young women exhibited aneuploid
conditions .
Battaglia D E et al , Hum Reprod 1997
The Assisted Conception Unit
The Assisted Conception Unit
Biological ageing of the egg is a physiological process
Oocyte aneuploidy
Embryonic aneuploidy
Mitochondrial DNA point mutations
The Assisted Conception Unit
EMBRYONIC ANEUPLOIDY
Aneuploidy in morphologically and developmentally
normal embryo markedly increases with maternal
age ( 13 - 53%) .
Harper J et al , Hum Reprod ; 1995
The Assisted Conception Unit
Biological ageing of the egg is a physiological process
Oocyte aneuploidy
Embryonic aneuploidy
Mitochondrial DNA point mutations
The Assisted Conception Unit
mtDNA Point Mutations
T414G point mutation in the control region of the mtDNA of
human oocytes :
4.4 % of oocytes of young patients ( 26-36 years)
vs
39.5 % in the older age group ( 37-42 years )
p<0.01
Barritt J ; Reprod Biomed 2000
The Assisted Conception Unit
Human donor ooplasmic transplantation ?
Cohen J et al ; Lancet 1997
The Assisted Conception Unit
Preimplantation genetic diagnosis in older patients
To biopsy or not to biopsy ?
The Assisted Conception Unit
PGD FOR ANEUPLOIDIES
The use of PGD in women of advancing age
could improve the success rate of IVF .
Gianaroli L et al , Fertil Steril 1999
Munne S et al , Hum Reprod 1999
The Assisted Conception Unit
Genetic Screening Of Embryos
Aneuploidy screen
60 cycles
Repetitive IVF failure ( RIVFF)
N: 30
Recurrent miscarriages (RM)
N: 10
Advanced maternal age > 39 (AMA) N: 7
The Assisted Conception Unit
Patient Selection Criteria
RIVFF: 3 or more failed IVF
RM : 3 or more miscarriages
AMA: > 40 years of age
The Assisted Conception Unit
Aneuploidy Screen
Examining chromosomes
– 13, 18, 21
– 15, 16, 22
– Monosomies
Live birth
Miscarriage
No implantation
Overall results from PGS cycles carried out on day 3 of embryo development
No. of couples
47
Average maternal age
37
No. Cycles to biopsy
60
No. oocytes
709, Average 11.8 ± 4.3
No. fertilised (2pn)
505
No. abnormally fertilised
122
No. of embryos biopsied
523, Average 8.7 ± 3.2
No. of embryos with result
Normal for chromosomes tested on biopsy
Cycles with more than 2 normal embryos
No. of embryos abnormal on biopsy
476 (91%)
85/476 (18 %), Average 1.4 ± 1
4/60
391/476 (82 %)
Cycles with ET
53
No. pregnancies
16
Pregnancy rate
per egg collection with biopsy
per embryo transfer
Ongoing
Pregnancy rate and maternal age groups
25-30 (6 cycles)
31-36 (18 cycles)
37+ (36 cycles)
27%
30%
26% (87.5% of initial pregnancies)
50% per embryo transfer, ongoing 83%
47% per embryo transfer, ongoing 94.5%
19% per embryo transfer, ongoing 100%
Overall results after PGS cycles and the follow up of non-transferred embryos according to referral reason
Referral group
AMA
RM
RIVFF
No. of couples
7
10
30
No. of cycles.
10
12
38
42.4
37.3
36
87, Average 8.7 2.75
110, Average 9.2 3.6
326, Average 8.6 3.2
77
96
302
25%
33%
29%
2
1
4
11 (14%) Average 1.1 0.7
17 (17.5%) Average 1.4 0.8
57 (19%), Average 1.5  1.3
65/76, 85.5%
76/93, 82%
212/269, 79%
Fully Chaotic mosaics
37/65, 57%
38/76, 50%
129/212, 61%
Other mosaic types
Aneuploid mosaic
Aneuploid/chaotic mosaic
diploid/aneuploid mosaic
Diploid/Chaotic mosaic
Other
22/65, 34%
36%
26%
8.7%
8.7%*
20.6%
29/76, 38%
41%
28%
7%
10%*
14%
79/212, 37%
29%
16%
11%
28%*
16%
Uniformly abnormal
6/65, 9.2%*
9/76, 12%*
4/212, 1.9%*
Embryos with meiotic errors
16/65, 25%*
20/76, 26%*
20/212, 9%*
Average maternal age
No. of embryos biopsied
No. of embryos with results
Pregnancy rate per ET
No. of cycles with no ET
Normal on biopsy
Result on follow up (abnormal)
The Assisted Conception Unit
RM and AMA embryos show consistent similarities: A three fold increase
in meiotic errors
↓
A common underlying mechanism in the causation of
aneulpoidy
One third of embryo abnormalities appear to have
started with a parental meiotic error.
Mantazaratou A et al; Hum Reprod 2007
The Assisted Conception Unit
Maternal meiosis is more error-prone compared
to paternal meiosis
↓
Most trisomies and/or monosomies detected in
prenatal samples and spontaneous abortions are
due to errors arising during the first maternal
meiotic division
Sherman SL, Human Molecular Genetics 1994
Hassold TJ, Environ Mol Mutag 1996
The Assisted Conception Unit
Maternal meiosis is more error-prone compared to
paternal meiosis
↓
The prolonged arrest in development that starts during
the fetal life ( at the dictyotene stage) and only end at
ovulation.
The Assisted Conception Unit
Study of individual chromosome in embryos with
errors → further similarities in the RM and AMA groups.
Most frequent meiotic abnormalities :
AMA group : Chromosome 21&18
RM group : Chromosome 21, 18 &13
Underlying common mechanism that links the infertility
in the two groups.
Mantazaratou A et al; Hum Reprod 2007
The Assisted Conception Unit
RIVFF embryos show a significantly lower incidence of
meiotic origin errors and an increase in post-zygotic
errors.
↓
Mitosis and not meiosis is more error prone in this group
Voullaire I et al ; Mol Hum Reprod 2002
The Assisted Conception Unit
RIVFF
Infertility is related to post fertilisation errors
↓
Independent of the parental meiosis
↓
Errors inherited by the embryo at the molecular level
The Assisted Conception Unit
RIVFF
Polymorphism (Pro 72) of the p53 tumour supressor
gene is associated with repeated implantation failure.
Kay C et al; Reprod Biomed Online 2006
The Assisted Conception Unit
The presence of a majority of fully chaotic embryos in all
groups of patients irrespective of age or reproductive
history.
↓
Genetic predisposition to generating chaotic embryos
The Assisted Conception Unit
Various degree of mosaicism in embryos does not
impair blastocyst formation.
Ruangvutilert P et al ; Prenat Diagn 2000
Magli MC et al; Hum Reprod 2000
Li M et al 2005; Fertil Steril 2005
30% of embryos from the RM group reached the
blasctocyst or morula stage → high implantation
potential → lack of progression in pregnancy
Outcome of 120 PGS cycles 2004-2006
• No. of cycles started
120
• Number of cycles to egg collection
• Clinical pregnancy rate per cycle started
101
25%
• Clinical pregnancy rate per egg collection
30%
• Clinical pregnancy rate per embryo transfer
33%
• ESHRE data VII - overall rate per ET
25%
The Assisted Conception Unit
The low frequency of embryos diagnosed as normal
(18%) (1.4 ± embryos/ cycle) has no adverse effect on
the pregnancy rate.
The pregnancy rate of 33 % per ET is above the
average of the latest ESHRE data for PGS :25% per ET
The Assisted Conception Unit
Follow up studies
Validation of Biopsy results
False positive rate : 0.6 % 1/354
All abnormal embryos on biopsy were abnormal on
follow up → varying degrees of abnormality.
The Assisted Conception Unit
Two cases:
Case 1-Youngest, age 26.
•
•
•
•
•
8 failed IVF attempts
12 embryos tested; results on 11
2 embryos normal for tested chromosomes transferred
Clinical pregnancy and normal birth
All remaining embryos confirmed abnormal
The Assisted Conception Unit
Case 2-oldest, age 44
• 2 previous PGS cycles-no other IVF
• 12 embryos tested; results on 10
• 3 embryos normal for tested chromosomes; 2
transferred ; all other confirmed abnormal
• Clinical pregnancy and live birth
( had 1:11 risk of DS, scans normal, no PND)
The Assisted Conception Unit
PGD FOR ANEUPLOIDIES SCREENING
Clinical Limitations
The success of IVF and PGD is very much dependant
on the number of eggs available .
A significant number of women > 40 have a limited
number of embryos and consequently PGD is not an
option .
The Assisted Conception Unit
PGD FOR ANEUPLOIDIES SCREENING
Technical Limitations
FISH : Number of chromosomes analysed in a single
cell is limited .
Mosaic embryos: less of a problem with blastocyst
biopsy
PGD FOR ANEUPLOIDIES SCREENING
Comprehensive Genomic Hybridisation ( CGH ) :
full analysis of all chromosome in a single
blastomere .
Wells D et al , Mol. Hum. Reprod ; 2000
Should Aneuploidy screen be offered be
routinely offered to infertile couples?
Mastenbroek study
• Women 35-41 yrs that planned IVF randomly assigned to undergo
3 cycles with embryo selection based either on PGS or on
morphology alone.
• None had previous failed IVF cycles.
• Single blastomere biopsied. Two embryos transferred.
• FISH in 2 rounds: Chroms. 1,16,17; then 13,18,21,X & Y.
i.e. not testing Chrs. 15 or 22.
Mastenbroek study
Results
• PGS group - 206 women. Control group - 202 women. Nearly 1/3
of women in both groups did not complete the intervention.
• Cumulative ongoing pregnancy rates:
PGS gp: 25% Control gp: 37% (p=0.01)
• Implantation rates:
PGS gp: 75/642 (11.7%) Control gp: 99/673 (14.7%)
Mastenbroek study
FISH Results
• Tested normal:
Abnormal:
Undiagnosed: 20.1%
38.4%
41.5%
• Comparative Implantation rates for PGD group:
Cycles with 2 ‘normal’ embryos transferred: 53/316 (16.8%)
Cycles with 2 undiagnosed embryos transferred: 6/100 (6%)
Mastenbroek study
Concerns
High % of undiagnosed embryos - suggests inexperienced group.
Omitting chromosomes 15 & 22 will lead to transfer of embryos abnormal for
these chromosomes.
Low pregnancy rates/cycle in control group - clinical 20%; ongoing 15%
Transfer of undiagnosed embryos affects implantation and pregnancy rates in
PGS group.
Top 10 monogenics
• Recessive
– Cystic Fibrosis
– β-thalassaemia
– Spinal muscular atrophy
– Sickle cell disease
• Dominant
– Myotonic dystrophy
– Huntington’s disease
– Charcot-Marie-Tooth disease
• Sex linked (specific diagnosis)
– Duchenne muscular dystrophy
– Fragile X syndrome
– Haemophilia
Inherited cancer predisposition
PND or PGD?
– Late onset
– Variable penetrance
– Variable expressivity
– Available treatments
– Prophylactic surgery
Retinoblastoma
RB1 - 13q14
Retinoblastoma
Osteogenic sarcoma
Pinealoma Leukemia
Lymphoma
Ewing sarcoma
Familial Adenomatous
polyposis
APC - 5q21
Colon carcinoma
Adrenal carcinoma
Thyroid papillary carcinoma
Periampullary carcinoma
Fibrosarcoma
Gastric adenocarcinoma
Medulloblastoma
Hepatoblastoma
Small intestine carcinoid
Desmoid tumor
Astrocytoma
Lynch syndrome
MSH2 - 2p22-2p21
MLH1- 3p21
Colon carcinoma
Endometrial cancer
Ovarian cancer
Renal cancer
Gastric cancer
Small bowel cancer
Liver cancer
Hereditary diffuse
gastric cancer
syndrome
CDH1 – 16q22
Gastric cancer
Mutiple endocrine
neoplasia type I
MEN I -11q13
Subcutaneous lipomas
Facial angiofibromas
Collagenomas
Pancreatic islet cell adenoma
Parathyroid adenoma
Pituitary adenoma
Adrenocortical adenomas
Prolactinoma
Glucagonoma
Insulinoma
Vasointestinal peptide tumor
Gastrinoma
Carcinoid tumors
Mutiple endocrine
neoplasia type 2b
RET - 11q11
Ganglioneuroma
Pheochromocytoma
Medullary thyroid carcinoma
Parathyroid disease rare
Tuberous sclerosis
TSC2 – 16p13
Myocardial rhabdomyoma
Multiple bilateral renal angiomyolipoma
Ependymoma
Renal carcinoma
Giant cell astrocytoma
Benign tumors - eye heart, lungs
Neurofibromatosis type I
NF1 – 17q11.2
Optic glioma
Meningioma
Hypothalamic tumor
Neurofibrosarcoma
Rhabdomyosarcoma
Duodenal carcinoid
Somatostatinoma
Parathyroid adenoma
Pheochromocytoma
Pilocytic astrocytoma
Tumors at multiple sites including CNS
Neurofibromatosis type 2
NF2 - 22q12
Meningioma
Glioma
Vestibular Schwannoma
Carney complex type 1
PRKAR1A - 17q23
Myxoid subcutaneous tumors
Adrenocortical nodular hyperplasia
Testicular Sertoli cell tumor, calcified
Pituitary adenoma
Mammary ductal fibroadenoma
Schwannoma
Psammomatous melanotic schwannomas
Thyroid carcinoma
Pheochromocytoma
Breast ovarian
cancer syndrome
BRCA1- 17q21
Breast cancer
Ovarian cancer
Pancreatic cancer
Endometrial cancer
Cervical cancer
Prostate cancer
BRCA2 – 13q12
Breast cancer
Ovarian cancer
Gastric cancer
Melanoma
Prostate cancer
Gall bladder
Bile duct
Pancreatic cancer
Von Hippel Lindau
VHL - 3p26
Pheochromocytoma
Hemangioblastoma,
Hypernephroma
Pancreatic cancer
Paraganglioma
Adenocarcinoma of
ampulla of Vater
Clinical
Nurse
PGD team
PCR team
FISH team
The Clinicians
The embryologists
http://www.conception-acu.com/
http://www.uclcentreforpgd.org/
The Assisted Conception Unit
The Assisted Conception Unit
Genetic screen for severe male factor infertility
Aneuploid karyotype :
13.7 % of men with azoospermia
4.6 % of men with oligozoospermia
The Assisted Conception Unit
Population based study : 165 couples TTP ( 12 months)
Group 1 (73 couples) : pregnancy 1-3 months
Group 2 (40 couples) : pregnancy 4-12 months
Group 3 (31 couples) : No pregnancy
DFI data from group 1 were significantly
different from group 2 (P<0.01) and from group
3( P<0.001)
No pregnancy with a DFI of ≥30 %
No correlation between DFI and semen parameters
Evenson DP et al ; Human Reprod 1999
The Assisted Conception Unit
Poor- quality sperm chromatin structure is highly indicative of
male subfertility regardless of conventional semen analysis
In vivo-fecundity deteriorate significantly when the DFI is
>30 %
Should Sperm DNA fragmentation test be integrated as a
complementary diagnostic tool for infertile patients ?
The Assisted Conception Unit
Should Sperm DNA fragmentation test be integrated as a
complementary diagnostic tool for infertile patients ?
Can sperm DNA damage be promutagenic?
Imprinting diseases
Cox GF et al ; Am J Hum Genet 2002
DeBaun MR at al ; Am J Hum Genet 2003
Childhood cancer
Fraga CG et al ; Mutat Res 1996
Ji BT et al ; J Natl Cancer inst 1997
Aitkens RJ et al ; Reproduction 2001
The Assisted Conception Unit
Biomarkers of implantation
HLA-G : type І human leucocyte antigen
* Produced by the syncytiotrophoblast.
Chu W et al ; Hum Immunology 1998
* Important role in immunoprotection of the semi-allogenic embryo
Fernandez N et al; Hum Reprod 1999
* sHLA-G has been identified in the culture media of 3-day-old embryos
Menicucci A et al; Hum Immunol 1999
The Assisted Conception Unit
Embryos derived from culture media expressing a high concentration of sHLA-G
exhibited :
* Accelerated cleavage
Menicucci A et al; Hum Immunol 1999
* High implantation rates
Fuzzi B et al ; Euro J of immunol 2002
Group A: ≤ 38 years (n=159)
A1 : sHLA-G
+(n=101)
Oocytes (n)
Fertilisation rate %
Mean no embryos
transferred
Clinical Pregnancy
rate%
Implantation rate
P-value
A2 : sHLA-G
- (n= 58)
Group B : 39-44 years (n=42)
B1: sHLA-G
+(n=29)
B2: sHLA-G
-(n=13)
626
82
348
77
174
71
97
73
2.9
3.5
3.2
2.9
22
52
71
38
<0.0001
9
25
15
5
<0.003
Sher G et al; RBM on line 2004
The Assisted Conception Unit
Low-dose aspirin significantly improves :
Ovarian responsiveness
Uterine and ovarian blood flow velocity
Implantation and pregnancy rates
Rubinstein M et al ; Fertil Steril 1999
Sperm Chromatin Structure Assay
sperm susceptibility to low PH- induced DNA denaturation
Population based study : 215 couples TTP ( 2 years )
DFI
cycles
≤8
332
8-11
319
11.3
0.671
0.421.08
12-19
326
13.5
0.939
0.601.08
.785
20-39 266
7.5
0.427
0.240.47
.004
≥ 40
1.7
0.129
0.020.97
.047
58
Fecundability
15.1
OR
95% CI
P value
1.000
.100
Spano M et al ; Fertil steril 2000
The Assisted Conception Unit
DFI prediction of fecundity is independent of semen
parameters
Spearman correlation coefficients : weak association of
DFI and semen parameters
-0.23 ( P<.0006) for DFI vs sperm concentration
-0.25 ( P <.0003) for DFI vs sperm morphology
Spano M et al ; Fertil Steril 2000
The Assisted Conception Unit
Superovulation and IUI
DFI< 27 %
Cycles
Clin preg
Live birth/cycle
DFI > 27 %
108
P-value
23
22 ( 20.2%)
19 ( 17.6%)*
1 (4.5%)
0.20
1 ( 4.5%)
NS
* One ectopic pregnancy
Bungum M et al ; Hum. Reprod 2004
The Assisted Conception Unit
Superovulation and IUI
119 patients underwent 154 cycles of IUI
Pregnancy rate : 8.4 % per cycle
10.9 % per patient
No samples with >12 % DFI resulted in pregnancy
Duran EH et al ; Hum Reprod 2002
The Assisted Conception Unit
In vivo-fecundity deteriorate significantly when the DFI
is >30 %
The Assisted Conception Unit
IVF and ICSI (21 patients)
DFI was significantly lower in the seven men that initiated
a pregnancy (15.4 ± 4.6 P=0.01)
High DFI ( 31.1 ± 3.2); n 14 : No pregnancy
Larson Kl et al; Hum Reprod 2000
The Assisted Conception Unit
IVF and/or ICSI
( 249 patients )
No difference in fertilisation between high and low DFI
groups
>30 % DFI : poor blastocyst rates( 30% P=.003 )
Increase in Spontaneous miscarriages in men with ≥ 30% DFI P=.11
No ongoing pregnancies (> 12 weeks ) in men with ≥ 30% DFI
Virro M et al; Fertil Steril 2004
The Assisted Conception Unit
IVF/ICSI
DFI< 27 %
Cycles
140
DFI > 27 % P-value
35
Clin preg /ET
47 ( 38.2%)
Live birth/cycle 44 ( 31.4%)
13 (38.2%)
12 ( 34.3%)
Bungum M et al ; Hum. Reprod 2004
NS
NS
The Assisted Conception Unit
IVF/ICSI
Two studies (n: 22 )
(n: 49)
Gandini L et al; Hum Reprod 2004
Seli E et al ; Fertil Steril 2004
No difference in clinical pregnancy rates
The Assisted Conception Unit
Blastocyst development
Significant negative correlation with % TUNEL positivity
When 20% was used as a cutoff for TUNEL positivity the % of
blastocyst development was 50 % higher in the <20%TUNEL –positivity
group
Seli E et al ; Fertil Steril 2004
The Assisted Conception Unit
Pre-treatment with metformin prior to IVF in patients with PCO
Retrospective study : 46 patients ( 60 IVF cycles)
Same amount of gonadotrophin
More mature oocytes ( 18 vs 13 )
Increased fertilisation rates ( 64 vs 43 % )
Higher clinical pregnancy rates ( 70 vs 30 % )
Stadtmauer LA et al ; Fertil Steril 2001
Prospective , randomised , double blind study : 73 patients
No significant differences in stimulation. oocytes or embryo parameters
The Assisted Conception Unit
AGE IS THE MAIN DETERMINANT OF
SUCCESS
BIOLOGICAL AGEING OF THE EGG IS
A PHYSIOLGICAL PROCESS THAT
CANNOT BE REVERSED WITH IVF
The Target : What part of the endometrium to aim for?
Coroleu et al Hum Reprod
2002; 17: 341
©
1.O
1.5
2.O
Randomized prospective
N = 180
Transabdominal U/S
Implantation rates
1.0 cm from fundus = 21 %
1.5 cm from fundus = 31 %
2.0 cm from fundus = 33%
The Target
Oliviera et al RBM Online
2004; 9: 435
©
Randomized prospective
N = 400 trs, Frydman
10%
21%
17%
10%
Transabdominal U/S
Implantation rates
Distance from fundus
relative to E.C.L.
< 40 %
41- 50 %
51- 60 %
> 60 %
=
=
=
=
10 %
17 %
21 %
10 %
The Target
Ribeirao Preto, Brazil
Oliviera et al
RBM Online 2004; 9: 435
©
17%
31%
Randomized prospective
N = 400 trs, Frydman
31%
13%
Transabdominal U/S
Ongoing pregnancy rates
Distance from fundus
relative to E.C.L.
< 40 %
41- 50 %
51- 60 %
> 60 %
=
=
=
=
13 %
31 %
31 %
17 %
The Target
Frankfurter et al
Fertil Steril 2003; 79: 1416
©
Retrospective, own control
N = 23 pairs, Wallace
NP
P
Transabdominal U/S video
Pregnancy v. Non-pregn.
Absolute distance from
fundus:
0.9 + 0.4 v. 0.7 + 0.4 cm
Relative distance from Cx:
0.66 + 0.13 v. 0.77 + 0.15
The Target
©