Veronica Bianchi

declares no conflict of interest

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

Morphological Data:slow freezing

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

Nottola 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

Clinical Data:slow freezing

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)

I PROTOCOL

0.1M sucrose 0.2M sucroseat freezing at thawing

(La Salle, 1985; Gook, 1993)

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.

Thawing cycles: oocyte cryopreservation data

Thawing cycles: clinical data

Thawing cycles: pregnancy outcome

The Italian IVF Law 40 - 2004

II PROTOCOL

0.3M sucrose 0.3M sucroseat freezing at thawing

(Fabbri et al 2001)

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)

Slow cooling: multicentric study• Borini et al. 2009

Biological and clinical outcome:

Slow cooling: multicentric study• Borini et al. 2009

Pregnancy outcome:

IV PROTOCOL

1 steps PROH (1.5M) + 0.2M sucrose 0.3M sucrose at freezing at thawing

(Bianchi et al 2007; submitted)

Our updated resultsUnpublished data

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

37

Thank you for your attention