The embryologists guide to advanced vitrification

The embryologists guide to
advanced vitrification
Joe Conaghan PhD
Los Angeles
April 21st 2011
Introduction
•
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•
•
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Importance of cryo in ART
Making good choices about what to vitrify
Devices and solutions
Open vs. Closed
Collapsing and hatching blastocysts
Can we do better?
Conclusions
Elective single embryo
transfer (eSET)
1. Ability to culture
embryos
2. Choice of embryos
for transfer and
cryopreservation
3. Reliable freezing
Fresh IVF Cycles: Number of embryos transferred
Day 3 ET
Day 5 ET
2007 CDC Data: Own oocytes only
Choosing your blastocysts
Common question
Do you freeze early blastocysts?
Blastocyst grading
Stage
ICM
Trophectoderm
Early
Good
Good
Average
Average
Poor
Poor
(cavity <50% vol.)
Blastocyst
(Cavity 50% or more)
Expanded
(Zona stretching)
Hatching
Can we agree on grading?
Stage:
ICM:
TE:
Can we agree on grading?
Stage:
Expanded
ICM:
TE:
Good
Good
Grading survey
Stage:
ICM:
TE:
119 Labs
participated
Results: Specimen 1
80
70
60
50
40
30
20
10
0
Morula
Early Blastocyst
Blastocyst
Expanded Blastocyst Hatching Blastocyst
Results: Specimen 1
80
70
60
50
40
ICM
30
TE
20
10
0
Good
Fair
Poor
Grading survey
Stage:
ICM:
TE:
119 Labs
participated
Results: Specimen 2
120
100
80
60
40
20
0
Morula
Early Blastocyst
Blastocyst
Expanded Blastocyst Hatching Blastocyst
Results: Specimen 2
70
60
50
40
ICM
30
TE
20
10
0
Good
Fair
Poor
Results: Day 3 testing
60
50
40
30
20
10
0
7
8
9
10
11
12
Results: Day 1 testing
120
107
100
80
60
40
20
2
0
0PN
1PN
2PN
9
1
3PN
4PN
Blastocyst quality
• Prefer to freeze embryos
that have a distinct ICM and
TE
• Early blastocysts may not
have clear differentiated cell
populations
• Pity freezes
Blastocyst quality
• Likely agreement on freezing good or
average
• Agree not to freeze poor embryos
• Disagree on relative importance of ICM and
TE
Which is the better blastocyst
1. Blastocyst with very nice ICM but only
average TE
2. Blastocyst with very nice TE but only
average ICM
Formation of the ICM
8-cell
16-cell
16-cell
32-cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell
Cell
O O
O I
O O
O I
O O
O I
O O
O I
O O
O O
OOOO
OOOO
O O
O O
O O
OOOO OOOO OOOI
12xO, 4xI
O O
I I
I I
OOOI
I I I I
I I I I
Average embryo should have 20-22 TE cells and 10-12 ICM cells
Moving from 32-cell to 64-cell, embryo can no longer
make ICM cells if they don’t already exist
Is trophectoderm more
important?
•Embryo puts more energy into making TE
•If ICM is poor, embryo likely doomed
•But TE relatively more important
Transfer embryos with:
good TE/average ICM or good ICM/average TE
Beyond the 32-cell stage
Trophectoderm cells can only
make trophectoderm
ICM cells may be able to make
trophectoderm, but only until the
64-cell stage
R. M. Schultz, Preimplantation embryo development, Molecular Biology in Reproductive Development 1999
Data from Marius Meintjes, personal commu
Data from Marius Meintjes, personal communication
Further reading
Richter KS, Harris DC, Daneshmand ST, Shapiro BS.
Quantitative grading of a human blastocyst: optimal
inner cell mass size and shape. Fertil Steril. 2001
Dec;76(6):1157-67.
Kovacic B, Vlaisavljevic V, Reljic M, Cizek-Sajko M.
Developmental capacity of different morphological
types of day 5 human morulae and blastocysts. Reprod
Biomed Online. 2004 Jun;8(6):687-94.
Criteria for vitrifying?
•Loose criteria for D5 blastocysts
•Tight control over D6 vitrification
•No ICM = no cryo
•Not keeping embryos until D7
•Assisted collapse used liberally
After transfer, embryos assessed by 2 embryologists
Early blastocysts can be
challenging
Is there a chance?
What are my chances?
…one out of a million.
So, there is a chance
So you’re telling me there’s
a chance
Yeeeeaaaahhhh
Borderline embryos
• Vitrifying poor embryos will hurt results
• If you can’t decide, you should preserve
• If your results are too good, you are being too
selective
• Recognize the importance of failure
Given the choice, patients likely choose freezing
Should we wait until D6?
D5 and D6 differences (OD)
Mean age = 43, n=177
35
30
25
20
15
Implantation rate (%)
10
5
0
D5
D5/D6
Implantation/transfer
D6
D5 only
D5+D6
D6 only
80/251 (32%)
8/33 (24%)
6/36 (17%)
Differences not significant (D5 vs. D6: p=0.07)
D5 and D6 differences
Mean age = 33, n=290, own oocytes
35
30
*
25
*
20
Implantation rate (%)
15
10
5
0
D5
Implantation/transfer
D5/D6
D6
D5 only
D5+D6
D6 only
104/318 (33%)
29/107 (27%)
29/128 (23%)
* p= 0.03
Choosing a device and protocol
Open vs. Closed
There is no authority that will
knowingly approve an open system
for storage of oocytes or embryos
Closed devices
Cryopette (Origio)
Rapid-i (Vitrolife)
Cryotip (Irvine Scientific)
Implementation (2007)
• Well trained staff
• Practice
• Vitrify good quality
embryos
• Artificial collapse?
• Assisted hatching?
Methods
• Vit Kit and
CryoTip
• Straws labeled
carefully
• One blastocyst
per straw
• Room temp.
Tip 1 : Examine straw carefully before starting
Materials and Methods
Vitrification Methods – Straw loading
1. Begin loading
immediately after
embryos in last drop
2. Medium to first mark,
then embryo to 2nd
mark
3. Continue loading
medium to 3rd mark
Materials and Methods
Vitrification Methods – Cooling procedure
We have settled on 8 mins
in ES for all blastocysts
Materials and Methods
Vitrification Methods – Cooling procedure
Materials and Methods
Vitrification Methods – Straw sealing
1. Seal small end and
check carefully
2. Seal large end and
check carefully
3. If not sure about seal,
reseal or load embryo
into a new straw
2nd mark
1st mark
Tip 2: Examine straw carefully
after sealing
Materials and Methods
Vitrification Methods – Storage
Must keep embryos in N2
at all times
Have system where label
can be read easily
Avoid shipping embryos
if possible
Methods continued
Straw warming
1. Check straw
label while
keeping straw
submerged
2. Quickly
transfer straw
to water bath
(370C)
Tip 3: Make sure you use a large
water bath. Stir straw in.
Ice formation during warming
00C
-1200C
-1960C
Methods continued
Straw unloading
1. Cut large end of straw
and gently attach
Hamilton syringe
2. Dry off fine end and
cut just above seal with
fine scissors
3. Gently expel contents
onto plate surface
Materials and Methods
Vitrification Methods – Warming Procedures
Materials and Methods
Vitrification Methods – Warming Procedures
Tip 4: Culture in medium with 20% DSS post warming
Blastocyst transfers in 2009
Who has embryos frozen?
Patient age
< 35 35-37 38-40 41-42 > 42
Number of Cycles
117
81
66
25
10
155
Number patients with
embryos to freeze (%)
Average number of
embryos frozen
94
(80)
56
(69)
32
(48)
15
(60)
2
(20)
139
(90)
4.3
3.7
3.6
4.5
1.0
6.0
Usable blastocysts
6.0
5.8
6.2
7.3
3.5
7.4
Overall 338/454 with embryos to freeze (74%)
1,630 Blastocysts frozen
Donor
Warming results: First 2 years
Patient age
<35
>40
OD
Cycles
164
80
76
21
217
Pregnancies
90
31
27
7
88
(mean)
55%
295
(1.8)
39%
145
(1.8)
36%
150
(2.0)
33%
51
(2.4)
41%
380
(1.7)
Sacs
112
35
35
10
120
24%
23%
20%
32%
Pregnancy rate
Emb. Transferred
Implantation rate 38%
35-37 38-40
When to warm and transfer
Natural
cycle
hCG
Controlled
cycle
Day or date
2PN’s
D3’s
D5 or D6
Mon
Day -1
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
P4
Day 1
P4
Day 2
P4
Day 3
P4
Day 4
P4
Day 5
P4
Day 6
P4
Day 7
Tue
Wed
Thur
Fri
Sat
Sun
Mon
Thaw
ET
Thaw/
ET
Warm/
ET
Blastocyst Recovery and Survival
Results 2007-2010
Cycles
Number of embryos
warmed
Number of embryos
recovered
Number of embryos
survived
776
1543
1482 (96%)
1374 (93%)
Implantation by stage
SET only, n = 182
60
b
50
a
40
30
a, b
20
10
0
EB
B
XB
HB
Early Blastocyst
Blastocyst
Expanded
Hatching
7/19
43/120
4/27
8/16
a, p = 0.04 and b, p = 0.03
Blastocyst survival
• Blastocysts look
very nice during and
immediately after
warming.
• Most ET’s done
within 1 hour of
warming
• Culture and ET in
20% SSS
Why does artificial collapse help?
1. Some embryos do not collapse
2. Reducing cavity allows direct CPA
access
3. Benefit may be from zona thinning
Blastocyst Survival Results 2007-2010
Overall
Cycles
Number of
embryos
warmed
Number of
embryos
survived
Not
Artificially
Collapsed Collapsed
776
576
177
1482
1143
288
1374
(93%)
1042a
(91%)
282a
(98%)
a p<0.05
Day 5 and Day 6 Survival
100%
Not Collapsed
Artificially Collapsed
98%
96%
63/64*
192/197*
94%
92%
90%
675/739
88%
200/225
86%
84%
Day 5
Day 6
* p < 0.05
SET Implantation Rates (n = 276)
50
Not Collapsed
Artificially Collapsed
45
8/17
40
35
30
25
5/13
13/37
47/144
12/36
20
15
10
4/29
5
0
Blast
Expanded Blast
Hatching Blast
Implantation rates <35
Overall
Cycles
Number of
embryos
transferred
Number of
embryos
implanted
Not
Artificially
Collapsed Collapsed
224
172
52
376
302
74
143
(38%)
106
(35%)
37*
(50%)
* p < 0.05
Implantation Results, <35
120
Day 5 Not Collapsed
Day 5 Artificially Collapsed
100
Day 6 Not Collapsed
80
Day 6 Artificially Collapsed
60
40
20
0
Survival
Implantation
Clinical Pregnancy
Clinical Pregnancy Rate
80%
Not Collapsed
70%
a
60%
Artificially
Collapsed
50%
40%
30%
20%
10%
0%
<35
35-37
38-40
>40
OD
a = sig. higher than non-AC group
Implantation Rates
60%
50%
Not Collapsed
a
a
Artificially Collapsed
40%
30%
20%
10%
0%
<35
35-37
38-40
>40
OD
a = sig. higher than non-AC group
Blastocyst Survival Rates - 2010
Overall
Cycles
Number of
embryos
warmed
Number of
embryos
survived
Not
Artificially
Collapsed Collapsed
289
124
165
487
220
267
448
(92%)
186
(85%)
262*
(98%)
* p < 0.05
D5 embryos do better
For patients <35 using own oocytes:
PR = 58% (51/88)
Mean of 1.8 embryos/FET
IR = 41% (62/153)
9 x twin, 1 x triplet (20% multiples)
Game plan: Freezing
1.
2.
3.
4.
5.
Aggressively vitrifying early blastocysts
Fairly “loose” in what we will vitrify
Collapsing any blastocysts that we can
Only one embryo/straw
Results continuing to improve
Game plan: Thawing
1.
2.
3.
4.
5.
Aim is to thaw and transfer 1
Young patients, D5 embryos, collapsed
Thaw 30-60 mins prior to FET
Culture and transfer in 20% SSS
Type of cycle not a concern
Outcomes of natural cycles vs. programmed cycles for 1677 frozen embryo
transfers.” Givens CR, Markun LC, Ryan IP, Chenette PC, Herbert CM, and
Schriock ED. Reprod Biomed Online, 2009 Sept, 19(3): 380-384
Where are we in 2011
1.
2.
3.
4.
5.
4 years experience
All 5 embryologists vitrifying and warming
Very loose on what we will vitrify
Collapsing most embryos
Still reducing the number of embryos
transferred
158 Blastocyst Transfers
Frozen cycle outcomes 2010
Patient age
< 35 35-37 38-40
> 40
Donor
Number of Transfers
46
23
16
5
68
Pregnancies (clinical)
59%
57%
25%
20%
44%
Embryos transferred
1.4
1.5
1.5
1.8
1.4
What are the problems that
people are having?
•
•
•
•
•
•
Not enough experience
Getting independent training
Using the device (straw sealing)
Use >500 ml water bath for warming
20% SSS in post warm culture media
Being aware of how quickly a straw will
warm
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