Blastocyst Stage Cryopreservation:
Toward Single Embryo Transfer Policy
Borut Kovačič
Izmir 2016
Dept. of Reproductive Medicine &
Gynecologic Endocrinology University Clinical Centre Maribor
Different approaches in IVF:
• How many embryos to transfer, how many to freeze?
• Which to transfer, which to freeze?
• When to transfer, when to freeze?
• How to freeze?
DAY 2
DAY 3
DAY 5
EMBRYOS FOR TRANSFER
ARRESTED EMBRYOS
SURPLUS EMBRYOS
Zhu et al., 2013
All warming cycles
1190 cycles of IVF/ICSI patients with surplus embryos on day-3
565 cycles with early embryo cryopreservation
Embryo vs. blastocyst vitrification
Zhu et al., 2013
Zhu et al., 2013
Blastocyst cryopreservation techniques
-150oC - 6oC
- 35oC +20oC
- 196oC Temp.
Time
Seeding
130 min -0.3oC/min.
PRINCIPLE OF SLOW FREEZING PRINCIPLE OF VITRIFICATION
+20oC
- 196oC Temp.
Time
T medium = - 70C osmolarity
T medium = - 300C osmolarity T medium = - 70C osmolarity T seeding baton = - 1960C induced crystalization
DEHYDRATION
DEHYDRATION
TEMP.
-1960C
TEMP.
-1960C
LIQUID MEDIUM
VITRIFIED MEDIUM (NO ICE CRYSTALS)
PRINCIPLE OF VITRIFICATION
Youssry et al., 2008
Slow vs. vitrification
Li et al., 2014
Li et al., 2011
SLOW VITRIFICATION
Slow vs. vitrification
Vitrification procedure
ES (10 min) VS (1 min) Loading onto holder
Plunge into LN2(-196ºC)
7.5% DMSO
+7.5% Ethylene glycol +20% DSS
15% DMSO
+15% ethylene glycol +0.5M sucrose
+20% DSS
Propanediol Ethylene glycol
Propanediol Ethylene glycol Ficoll
Warming procedure
TS (37ºC)(1 min) DS (4 min) WS-I (4 min) WS (4 min)
1.0M Sucrose 20%DSS
0.5M Sucrose
20%DSS 20%DSS 20%DSS
• Electron microscope grid
• Open pulled straw
• Cryoloop
• Cryotop
• Cryotip
• Cryoleaf (McGill)
• Cryo Bio System straw
Vitrification carriers
Close vs. open
2.500ºC/min vs. 23.000ºC/min Sterile vs. non-sterile
2.500ºC/min vs. 23.000ºC/min Sterile vs. non-sterile
Close vs. open vitrification
Iwahata et al., 2015
2.500ºC/min vs. 23.000ºC/min Sterile vs. non-sterile
Close vs. open vitrification
Chen et al., 2013
Early (compact) vs. Expanded blastocysts
• Compact stages survive cryopreservation better than expanded stages (Tao et al., 2001).
• Cryopreservation altered the quantitative bioenergy/redox parameter at the greater extent in the morulae than in the blastocysts (Somosko et al., 2015).
• Day-5 only (morulae & blastocysts) cryopreservation will increase the workload in the lab and decrease success rate per thawing
• Day-5/6 blastocyst (expanded blastocyst) cryopreservation is more recommended approach.
Day-5 vs. Day-6 blastocysts
Optimal vs. suboptimal blastocysts
Maribor results MSRM, 2015
Day-5 vs. Day-6 blastocysts Single vs. double blastocyst
Kang et al., 2013
Artificially collapsed vs. intact blastocysts
Vanderzwalmen et al., 2002
Micro-needle puncture
Control Son et al. 2003 Micro-needle
puncture
Hiraoka et al., 2004 Micropipetting Zech et al., 2005 Spontaneous
hatching
Assisted (Mechanically) Mukaida et al., 2006 Laser pulse
Mukaida et al., 2006 Microneedle puncture Laser pulse Iwayama et al., 2011 Laser pulse Osmotic shock Gala et al., 2014 Laser pulse Control
Cao et al., 2014 Laser pulse Microneedle puncture
Kader et al., 2009
Van Landuyt et al., 2015
Collapsed blastocysts
(N = 118)
Intact blastocysts
(N = 119)
P
Intactness of blastocysts
Fully intact blastocysts, n (%) 97 (82.2) 90 (75.6) NS Partly damaged blastocysts, n
(%)
21 (17.8) 28 (23.5) NS Not vital blastocyst, n (%) 0 (0) 1 (0.9)
ICM
Grade A, n (%) 45 (38.1) 40 (33.6) NS
Grade B, C, n (%) 73 (61.9) 78 (66.1) NS
TE
Grade A, n (%) 40 (33.9) 34 (28.6) NS
Grade B, C, n (%) 78 (66.1) 84 (71.2) NS
Results: Morphology at transfer
Maribor results ESHRE, 2015
Collapsed blastocysts
(N = 118)
Intact blastocysts
(N = 119)
p
Pregnancies, n (%) 39 (33.1 ) 38 (32) NS Miscarriages, n (%) 8 (20.5) 5 (10.5)
Live births, n (%) 31 (26.3) 34 (28.6) NS Live births / op8mal blastocyst, n (%) 22 (41.5) 20 (40.5) NS
Results: Transfer outcome
Maribor results ESHRE, 2015
B1
B2
B3
B4
B5
B6
B7
B8 Vitrification of all surplus blastocysts scored from B1-B6.
Vitrification on day 5 or on day 6.
BLASTOCYSTS FOR VITRIFICATION Scoring of day 5 morulae and blastocysts (Kovačič et al., 2004; RBMOnline)
Selection of blastocysts for cryopreservation
B1
B2
B3
B4
B5
B6
B7
B8 BLASTOCYSTS FOR VITRIFICATION
Scoring of day 5 morulae and blastocysts (Kovačič et al., 2004; RBMOnline)
Selection of blastocysts for cryopreservation
A
B
Survival and reexpansion after thawing
A
B
C
D Kovacic & Vlaisavljevic, InTech, 2010
Fresh vs. vitrified blastocysts
Ku et al., 2012
Vitrified-warmed BT cycles resulted in statistically significantly higher CPR and IR compared with fresh BT cycles.
Zhu et al., 2011
30%
70%
Destiny of blastocysts from 404 cycles with cryopreservation in year 2008
Transferred fresh (n=620)
Cryopreserved (n=1429)
35%
8%
57%
Destiny of cryopreserved blastocysts (n=1429) from 404 patients in 5 years period
Transferred after thawing (n=495) Damaged after thawing (n=117) Remained unused after 5 years period (n=817)
Cumulative outcome of fresh and related cryo cycles
(Maribor statistics)
from 202 patients
Cumulative delivery rate from fresh and thawing cycles in a 5 years period
0,0 10,0 20,0 30,0 40,0 50,0 60,0 70,0
50,0 50,0 50,0 50,0 50,0 50,0 6,4 10,6 12,3 13,1 13,3 1,2 1,9 2,3
65.6%
Cumulative delivery rate
Deliveries from thawed blc (after succesful fresh cycle)
Deliveries from thawed blc (after unsuccesful fresh cycle)
Deliveries from fresh blc
Proportion of „freeze all“ cycles and prevalence of OHSS
0 1 2 3 4 5 6 7
2011 2012 2013 2014 2015
OHSS
To freeze all
% of cycles
„To freeze all“ cycles
N
• Antagonist cycles
• Risk for OHSS
• Ovulation trigerring with agonists (NOT with hCG)
„To freeze all“ cycles
ET 1 ET 2 ET 3 ET 4 ET 5 ET 6 No/Total No. 116/123 48/53 22/27 12/13 5/7 1/2 Cumulative
clinical PR
62
(53.4%)
79
(68.1%)
85
(73.3%)
89
(76.7%)
90
(77.6%)
90
(77.6%)
Of 33 non-pregnant, 22 (66.7%) still had embryos left.
„To freeze all“ cycles
42,2%
62,1% 69,0% 72,4% 73,3% 75,9%
0,0%
10,0%
20,0%
30,0%
40,0%
50,0%
60,0%
70,0%
80,0%
90,0%
100,0%
1. cycle 2. cycle 3. cycle 4. cycle 5. cycle 6. cycle
Hundreds
Cumulative delivery
Conclusions
• Vitrification became a standard in cryopreservation of blastocysts.
• It is a simple method resulting higher survival and implantation rates compared to slow freezing.
• Prolonged embryo culture and efficient cryopreservation (vitrification) of surplus blastocysts offer:
– Good posibilities for elective single blastocyst transfer.
– Easier decision for the cancellation of transfer in prevention of OHSS.
• There is not enough evidence to make a conclusion about the superiority of:
– Specific cryoprotectant – Specific embryo carrier – Open or close vitrification – Artificial shrinkage
Figure 1
Reproductive BioMedicine Online 2013 27, 310-315DOI: (10.1016/j.rbmo.2013.05.016)
Maribor approach
Knez et al., 2013 Fresh cycles
Figure 2
Reproductive BioMedicine Online 2013 27, 310-315DOI: (10.1016/j.rbmo.2013.05.016) Knez et al., 2013 Fresh cycles
Figure 3
Reproductive BioMedicine Online 2013 27, 310-315DOI: (10.1016/j.rbmo.2013.05.016) Knez et al., 2013 Fresh cycles
0 10 20 30 40 50 60 70 80
2008 2009 2010 2011 2012
69,8 72,2 73,7 70,5 71,8
55,8 56,2
47 43,9
52,7
28,3 27,9
21,1 18,1
25
Cycles with >2 embryos Cycles with blastoc for cryo
Embryos developed to BC for cryo
Fresh cycles with surplus embryos (cycles with >2 embryos developed)
%
Fresh cycles
(with cryopreserved blastocysts)from year 2008 and related thawing cycles in the following 5 years
period
0 50 100 150 200 250 300 350 400 450
404
119 113
55 36
7 8
+338
Short vs. prolonged embryo culture
Early embryos
+ ET before embryonic genome
activation
+ Short exposure to non-natural conditions
+ No loose of embryos due to suboptimal conditions
+ More safe for embryos
Blastocysts
+ Better selection of embryos for ET + Self selection
+ No need for detailed morphology assessment
+ Higher implantation rate + Better synchronisation with
endometrium
+ Allows single embryo transfer + Reduction of number of
cryopreserved embryos - Selection on the basis of low
predictive markers
- Multiple embryo transfer - Huge cryo programme
- Increased number of thawing cycles - Higher cost
- Culture after genome activation - Lost of embryos due to suboptimal
culture
- Possible cancellation of transfer - Possible effect on epigenetics
Cryopreservation methods:
• Vitrification from 2008 (n = 650-700 warming cycles per year):
– Irvine Scientific (Ethylene glycol + DMSO) vitrification media.
– CBS straws
• Mean nuber of transferred: 1.5 blastocysts.
– Criterium for survival after thawing: >50% of intact cells.
• Transfer in:
– spontaneous cycles in ovulatory patients (urine LH test),
– artificial cycles (estradiol valerate (Estrofem) and progesterone (Utrogestan)) in anovulatory patients and some patients from abroad.
• Ultrasound measurement of endometrial thickness from day 10.
• Enrolment:
- patients having vitrified blastocysts from both groups - intended for single blastocyst transfer (SBT)
• Randomisation by alternation (at warming) once collapsed,
once intact blastocyst
• Analysis of blastocyst reexpansion and clinical outcome
Thawing cycles N=237
Collapsed BC N=118
Intact BC N=119 Time-lapse Time-lapse
SBT SBT
Maribor results ESHRE, 2015
Artificially collapsed vs. intact
blastocysts
Single blastocyst transfer
Double blastocyst transfer
B l a s t o c y s t quality
Optimal Non-optimal Optimal Optimal
Optimal Non-optimal
Non-optimal Non-optimal
No. of ET 330 499 177 128 311
Clin. preg. 142 (43.0) 142 (28.5) 72 (40.7) 40 (31.2) 72 (23.2) Deliveries 109 (33.0) 50 (18.2) 57 (32.2) 29 (22.7) 49 (15.6) Twins 0 1 (2.0) 16 (28.1) 6 (20.7) 11 (22.4)
Outcome of single and double transfers of vitrified blastocysts
Maribor statistics
Not optimal laboratory or cryoprogramme
Double or multiple embrotransfer
Good laboratory and cryoprogramme Single embrotransfer
