Post on 26-Dec-2015
transcript
Pre & Post morphological selection of oocytes cryopreserved with slow freezing
clinical outcomes
Veronica Bianchi Ph.D. ESHRE Senior Embryologist
Outlines
• Indications
• Morphological evaluation (using slow freezing)
• Protocols and procedures available in the literature
• Clinical outcome worldwide
• Our results with slow cooling
• Conclusions
o Restrictions to embryo storage Ethical Legal
o Failure to produce semen sample and/or no sperm the day of egg retrieval
o Fertility preservation in cancer patients
o Preservation of women’s fertility
o Egg donation
Indications
Goals of Cryopreservation
• Minimize/prevent cryo-injuries from– Ice crystals (internally and externally)– Toxicity of cryoprotectants– Solute effects– Osmotic shock– Chilling sensitivity– Fracture damage
• Preserve structural integrity and biological viability of cells
Slow freezingSlow freezing
- 160
- 140
- 120
- 100
- 80
- 60
- 40
- 20
0
20
40
0 15 30 45 60 75 90 105 120
Time (min)
Tem
pera
ture
°C
• Wash in PBS
• 1.5 M Pr-OH, 10 min
• 1.5 Pr-OH / 0.2 M sucrose
• load
• RT to -8°C, -2°C/min
• Seed
• Hold, 10 min
• -8°C to -30°C, -0.3°C/min
• -30°C to -150°C, -50°C/min
• Plunge into LN2
sucrose
H2OH2O
Pr-OH
Strict adherence to protocolStrict adherence to protocol
Handling Incubation times
Loading in straws
1.5 M PROH, 0.1/0.3M sucrose in PBS (L)
1.5 M PROH in PBS (S)
5 min10 min
Oocyte(s)
mediumair
• RT, 30 sec
• 30°C water, 40 sec
• RT,1.0 M Pr-OH, 0.3 M sucrose, 5 min
• RT, 0.5 M Pr-OH, 0.3 M sucrose, 5 min
• RT, 0.2 M Pr-OH, 0.3 M sucrose, 10 min
• RT, PBS, 10 min
N.B. All freezing/thawing solutions prepared with PBS supplemented with PPS 20% (v/v)
Rapid thawingRapid thawing
Spindle and chromosome organization in human oocyte frozen after exposure to
1.5 Pr-OH / 0.1 M sucrose or 1.5 Pr-OH / 0.3 M
Coticchio et al., Hum Rep 2006
Title0.2M/0.3M Protocol Resulted in Less Ultrastructural Damage
––+MII spindle damage
+++Cortical granules loss
++ ++ Ultrastructural damage (vacuolisation)
0.2M/0.3M0.3M/0.3M0.1M/0.2M
Protocols
Coticchio et al., 2006; Nottola et al., 2007; 2008; 2009; De Santis et 2008; Cobo et al., 2008; Bromfield et al., 2009; Coticchio et al., 2009 Bianchi et al 2007
1980 1990 2000
200719971986
Steady outcome improvement by slow freezing over time
2001-2006
Survival 20-30% 40-50% 70-75% 70-75%
Fertilization <40% 40-50% 70-75% 70-75%
Cleavage (4-cell)
??? ??? 10-15% 20-30%
Implantationper thawed oocyte
??? ??? 2.5-2.7% > 5%
PROH-Sucrose – SLOW FREEZING protocol
4 different protocols
a) 1.5M PROH + 0.1M sucrose (La Salle, 1985; Gook, 1993) b) 1.5M PROH + 0.3M sucrose (Fabbri et al., 2001) c) 0.75 M PROH and 1.5M PROH + 0.2M sucrose (Borini et al. 2009)
d) 1.5M PROH + 0.2M sucrose (Bianchi et al. 2007)
Materials and methods:We started our program on January 1997 until December 2000.68 couples which asked not to have supernumerary embryos were involved in the program.
THREE of the recovered oocytes were inseminated, the remaining oocytes were cryopreserved.
Enough oocytes were thawed in order to obtain 3-4 oocytes suitable for ICSI.
TitleResults from Different Centres Using 0.3 M Protocol were Similar
Borini
2006
Levi Setti
2006
La Sala
2007
De Santis
2007
% Survived 74 69 73 71
% Fertilised 76 68 N.R. 80
% Implantation 5.2 5.8 3.4 5.7
% Implantation
per thawed oocyte 2.6 1.9 2.5 2.6
III PROTOCOL
2 steps PROH (0.75M - 1.5M) + 0.2M sucrose 0.3M sucrose at freezing at thawing
(Borini et al., 2010)
IV PROTOCOL
1 steps PROH (1.5M) + 0.2M sucrose 0.3M sucrose at freezing at thawing
(Bianchi et al 2007; submitted)
Overall survival, fertilization and cleavage rates
No patients (at least one thawing cycle) 342
Mean age (DS) 35,44.28
No. of thawing cycles 443
No. of oocytes
Thawed 2458
Survived (%) 1766 (71.8)
Microinjected 1296
2PN (%) 1010 (77.9)
Cleaved (%) 955 (94.5)
G1 (%) 339 (35.5)
G2 (%) 461 (48.3)Bianchi et al. submitted 2011
No. of embryo transfers 394
No. of embryos transferred 907
No. pregnancies 90
Pregnancy rate/embryo transfer (%)
90/394 (22.8)
Pregnancy rate/thawing cycle (%) 90/443 (20.8)
Pregnancy rate/patient (%) 90/342 (26.3)
Multiple pregnancy rate (%) 26/90 (28.9)
No. of gestational sacs 122
No. of FHB 93
Implantation rate 13.5
Implantation/injected oocyte rate 9.4
Abortion (%) 32 (35.5)
Overall clinical data
Bianchi et al. submitted 2011
Survival, fertilization and cleavage rates
Bianchi et al. submitted 2011
≤34 35-38 ≥ 39
No patients ( at least one thawing cycle) 138 114 90
Mean age (DS) 31.1 4.28 36,44.28 40.64.28
No. of thawing cycles 177 148 118
No. of oocytes
Thawed 993 815 650
Survived (%) 737 (74.2) 570 (70.0) 459 (70.6)
Microinjected 515 435 346
2PN (%) 410 (79.6) 347 (79.8) 253 (73.1)
Cleaved (%) 382 (93.2) 331 (95.4) 242 (95.6)
G1 (%) 133 (34.8) 105 (31.8) 102 (42.2)
G2 (%) 186 (48.7) 169 (51.0) 106 (43.8)
≤34 35-38 ≥ 39
No. of embryo transfers 155 131 108
No. of embryos transferred 365 301 241
No. pregnancies 43 28 19
Pregnancy rate/embryo transfer % 27.7 21.4 17.6
Pregnancy rate/thawing cycle % 24.3 18.9 16.1
Pregnancy rate/patient % 31.2 24.6 21.1
Multiple pregnancies (%) 16 (37.2) 4 (14.3) 6 (31.6)
No. of gestational sacs 61 35 26
No. of FHB 49 24 20
Implantation rate % 16.7 11.6 10.8
Implantation/injected oocyte % 11.8 8.0 7.5
Abortion (%) 10 (23.3) 12 (42.8) 10 (52.6)
Overall clinical data
Bianchi et al. submitted 2011
Impact of protocol: Performance in vitro of embryos from
sibling fresh and frozen oocytes
0
5
10
15
20
25
30
35
404-
cell
emb
ryos
at
44-
46 h
p.i
.
Fresh 0.3 - 0.3 Cook 0.2 -0.3
Freezing protocolP < 0.001 for all protocols in comparison to control
Borini et al., unpublished