Download - Thermo Fisher Scientific

Real experiences with PGS and transition
to NGS
Carmen Rubio
Valencia, Spain
Corporate presentation 2013
PGS: technology evolution
day-3 biopsies
2 blastomeres
1991-1995
PCR
2008
FISH
Blastocysts
Deferred
transfer
2012
2010
2014
Array Comparative qPCR
Genomic
Hybridization
(aCGH)
SNP
microarray Next-Generation
Analysis of an identified single gene disease
2-12 chromosomes
24 chromosomes
Sequencing
(NGS)
24 chromosomes
+
Mit DNA; mutations
aCGH vs. FISH
aCGH vs. NGS
N= 6,934 aCGH vs. 284 NGS cycles
% Abn
80
70
60
50
40
30
20
10
0
PR/Tr
IR
MR
aCGH
NGS
Agrees with published data
PR: 74.7% NGS
vs.
69. 2% aCGH
NGS Studies
NGS: day-3 and blastocyst analysis
Now?
Future?
 Blastomere and trophectoderm samples
 Mitochondrial DNA analysis
 Whole-chr and segmental aneuploidies
 Embryo viability markers
 Single gene disorders
Personalized
Reproductive
Medicine
NGS platform
Ion Torrent™ Technology
Up to 24 samples
loaded on a chip
Ion PGM™ System
Changes in pH
detected
Sequencing
Ion ReproSeq™ System
PGS-NGS data
Euploid embryo
Mon 20
Tris 22
NGS concordance with aCGH
Embryo aneuploidy
NGS
aCGH
Evaluation of PGS-NGS
PGS-NGS evaluation
Aneuploidy analysis
(Whole-aneuploidies)
Cell lines
Single
Pool
N=42
CR 95.5%
N=38
CR 100%
N=81
CR (per embryo) = 97.5%
CR (per chr) = 99.9%
Cell line
Karyotype
JV
46, XY
AG02102
46, XX
GM 00563
45, X0
GM 09286
47, XY, +9
Evaluation of PGS-NGS
Aneuploidy analysis
(Whole-aneuploidies)
N=133
CR (per embryo) = 99.2%
CR (per chr) = 99.9%
N=61
CR (per embryo) = 97.2%
CR (per chr) = 99.9%
PGS with aCGHand NGS
Day 3 samples
Blastocyst samples
6,934 aCGH
335 aCGH
284 NGS
60 NGS
NGS technology
Day-5 /Day-6 transfer
PGS: To whom ?
RM
RIF
MF
PTP
AMA
<38yrs
<38yrs
<38yrs
<38yrs
38-44
Mean Age
34.8
34.9
33.9
35.3
40.8
Abnormal embryos
68.3
66.5
66.1
68.9
86.3
Embryo transfers
75.5
80.7
78.3
85.2
40.0
PR/transfer
56.7
54.4
59.2
65.4
51.9
Implantation rate
48.0
46.1
53.3
57.9
47.6
PR/cycle
42.8
44.0
46.2
55.7
20.8
Miscarriage rate
11.9
9.8
10.0
14.7
9.8
Day-3 vs. blastocyst?
Percentage of cycles with euploid embryos (7,269 cycles)
6,934 cycles with day-3 biopsies
335 cycles with day-5/6 biopsies
Tr D3
100
90
80
70
60
50
40
30
20
10
0
Tr D5
aCGH: abnormal embryos
Percentage of abnormal embryos (38,031 embryos) analyzed
36,281 embryos with day-3 biopsies
1,750 embryos with day-5/6 biopsies
Tr D3
120
100
80
60
40
20
0
Tr D5/6
aCGH detection rates
VS.
Day-3
Blastocyst
Mean age
39.5
37.6
Mean MII
9.6
13.1
Mean analyzed
5.2
5.4
% abnormal embryos
81.0
54.4*
% chaotic pattern
16.1
2.1*
% complex aneuploidy
18.6
6.2*
% partial aneuploidies
5.4
6.1
(*p<0.05)
aCGH: Day-3 vs. blastocyst biopsies
80
Day-3 biopsies:
N= 6,934 cycles
(36,281 embryos)
PR/transfer
11.7 MII
9.1 MII
7.4 MII
60
IR
PR/cycle
40
20
0
<38 yrs
80
Day-5/6 biopsies:
N= 335 cycles
(1,750 embryos)
13.8 MII
38-42
12.8 MII
43-46
8.2 MII
60
PR/transfer
IR
PR/cycle
40
20
0
<38 yrs
38-42
43-46
NGS: Day- 3 vs. Blastocyst
NGS: 284 day-3 vs. 60 blastocyst cycles
% Abn
PR/Tr
IR
MR
80
60
40
20
0
Day-3
Blastocyst
NGS and aCGH inform of similar % abnormal embryos, pregnancy and
implantation rates, and miscarriage rates in NGS for blastocyst biopsies.
Few cycles included.
NGS: day3 data
NORMAL
ABNORMAL
CHAOTIC
BLOQ
MC
BT
4.1
6.0
13.4
18.3
20.0
21.7
56.4
22.1
11.4
BC
BE
BH
17.7
2.7
3.6
18.0
2.5
1.3
6.9
0.3
2.8
52.0
90.0
21.7
61.5
0
21.4
BHi
Total Blast
(*p<0.05)
Blastocyst biopsies
% Cycles
Transfer type
Arrested before biopsy
11%
Fresh
All Day-5 biopsy
11%
Fresh
Day-5 and day-6 biopsy
50%
Defered
All Day-6 biopsy
28%
Defered
Mercader, IVI-Valencia
Vitrification at different stages
Fresh
Ovo Vit
D2 Vit
D3 Vit
Blast
No. CYCLES
617
78
162
454
163
MEAN AGE
39.1a
40.6a
39.5
39.2
37.7
MEAN No. OF MII
9.3b,c
9.1
11.4b
9.7
13.3c
No. TRANSFERENCES
332
35
98
266
74
No. PREGNANCIES (%)
53.3
48.6
55.1
52.2
62.2
IMPLANTATION RATE
48.9d
47.6
46.8
46.7
59.3d
1.4
1.3
1.5
1.3
1.4
No. ANALYZED EMBRYOS
3110
351
955
2673
758
No. INFORMATIVE EMBRYOS (%)
98.5
97.1
98.7
98.7
97.5
No. ABNORMAL EMBRYOS (%)
79.6e
83.6
77.8
81.4
56.8e
MEAN TRANSFERED EMBRYOS
Mercader et al., 2016
Oocyte vitrification
2 ciclos
Nº de pacientes (%)
3 ciclos
4 ciclos
73 (57,6) 37 (28,5) 14 (10,8)
5 ciclos
6 ciclos
4 (3,1)
2 (1,5)
Pacientes
≥1 embrión normal
Embrión normal 1º ciclo (%)
40
12
10
2
0
21 (52,5)
5 (41,6)
3 (30,0)
0
0
Embrión normal 2º ciclo (%)
19 (47,5)
3 (25,0)
1 (10,0)
1 (50,0)
0
Embrión normal 3º ciclo (%)
-
4 (33,3)
4 (40,0)
0
0
Embrión normal 4º ciclo (%)
-
-
2 (20,0)
1 (50,0)
0
Embrión normal 5º ciclo (%)
-
-
-
0
0
Embrión normal 6º ciclo (%)
-
-
-
-
0
aCGH: day-3 biopsy RCT
Study groups
 Advanced maternal age (38 - 41 years)
 Severe male factor (≤ 2 million/mL)
Polar Body
biopsy
Day-0
Cleavage stage
biopsy
Blastocyst
biopsy
Day-1
ClinicalTrials.gov NCT01571076
AMA - RCT with aCGH on day-3 biopsies
VS.
38-42 years
Blastocyst
PGS
P-value
No. of cycles performed
110
102
---
39.4 (1.1)
39.5 (1.1)
NS
Percentage of transfers
99.1
66.7
p<0.0001
Mean embryos/transfer
1.8 (0.6)
1.3 (0.7)
p<0.0001
48
38
---
No. of miscarriages (%)
23 (47.9)
1 (2.6)
p<0.0001
Delivery rate/transfer
22.9
54.4
p<0.0001
Delivery rate/cycle
22.7
36.3
p=0.0349
Ongoing IR
14.9
46.7
p<0.0001
Liveborns/cycle
26.4
42.1
p=0.02
Mean age (SD)
No. of pregnancies
Two-side Fishers´ test
ClinicalTrials.gov NCT01571076
ASRM 2015
MF - RCT with aCGH on day-3 biopsies
VS.
Blastocyst
CCS
P-value
41
44
---
32.6 (3.4)
33.0 (2.8)
NS
97.6
88.6
NS
1.9 (0.6)
1.5 (0.7)
P=0.0079
23
29
---
6 (26.1)
1 (3.4)
P=0.0237
Ongoing PR/transfer*
42.5
71.8
P=0.0078
Ongoing PR/cycle*
41.5
63.6
P=0.0334
Ongoing IR
22.7
55.2
P=0.0001
No. of cycles performed
Mean age (SD)
Percentage of transfers
Mean embryos/transfer (SD)
No. of pregnancies
No. of miscarriages (%)
*12 weeks ongoing pregnancies
* p<0.05 T-Student and one-side Fishers´ test
ASRM 2015
Acknowledgements
Scientists
Technicians
Carlos Simón
Akinwole Adedoyin
Ayala Gustavo
Bermell Soledad
Centelles Vicente
Darvin Tristan
Escobedo Milagros
Eskridge Roderick
Al-Assmar Nasser
Alberola Trini
Blesa David
Bandeira Carla
Campos-Galindo Inma
Cervero Ana
Chopra Rupali
Cinnioglu Cengiz
Coprerski Bruno
Chiu Yatfung
Díaz-Juan Antonio
Díaz Patricia
Dizon Bautista Abelard
Elshaikh Noon
Garcia-Herrero Sandra
Gómez Eva
Jiménez Jorge
Kayali Refik
Khajuria Rajni
López Pilar
Mae Hoover Larissa
Marín Carlos
Marín Lucía
Martín Julio
Martínez José Antonio
Mateu Emilia
Milán Miguel
Mir Pere
Miravet José
Moreno Inma
Navarro Roser
Peinado Vanesa
Pinares Ania
Poo María Eugenia
Riboldi Marcia
Rincón Alejandro
Rodrigo Lorena
Rodríguez Beatriz
Rubio Carmen
Ruiz María
Sánchez Maribel
Sanz Lucía
Sen Gurkan
Uehara Mariane
Valbuena Diana
Vera María
Vilella Felipe
Whittenburg Alex
García Mirian
Gómez María
Martinez Asun
Martinez Lucia
Martinez Sebastian
Martinez Tantra
Mateos Pablo
Moles Sara
Morata María Jesus
Nguyen Tuan
Nieto Jessica
Peris Laura
Pozo Ana
Singh Vinita