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

oRestrictions to embryo storage Ethical Legal oFailure to produce semen sample and/or no sperm the day of egg retrieval oFertility preservation in cancer patients oPreservation of women’s fertility oEgg 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 freezing Time (min) Temperature °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 LN 2 sucrose H2OH2O H2OH2O Pr-OH

Strict 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) medium air

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 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

Title 0.2M/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

Steady outcome improvement by slow freezing over time Survival20-30% 40-50% 70-75% Fertilization<40% 40-50% 70-75% Cleavage (4-cell) ??? 10-15% 20-30% Implantation per thawed oocyte ??? % > 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 sucrose at freezing at thawing (La Salle, 1985; Gook, 1993)

Materials and methods: We started our program on January 1997 until December 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

II PROTOCOL 0.3M sucrose at freezing at thawing (Fabbri et al 2001)

Title Results from Different Centres Using 0.3 M Protocol were Similar Borini 2006 Levi Setti 2006 La Sala 2007 De Santis 2007 % Survived % Fertilised7668N.R.80 % Implantation % Implantation per thawed oocyte

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 Biological and clinical outcome:

Slow cooling: multicentric study Borini et al 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 results Unpublished data

Overall survival, fertilization and cleavage rates No patients (at least one thawing cycle)342 Mean age (  DS)35,4  4.28 No. of thawing cycles443 No. of oocytes Thawed2458 Survived (%)1766 (71.8) Microinjected1296 2PN (%)1010 (77.9) Cleaved (%)955 (94.5) G1 (%)339 (35.5) G2 (%)461 (48.3) Bianchi et al. submitted 2011

No. of embryo transfers394 No. of embryos transferred907 No. pregnancies90 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 sacs122 No. of FHB93 Implantation rate13.5 Implantation/injected oocyte rate9.4 Abortion (%)32 (35.5) Overall clinical data Bianchi et al. submitted 2011

Survival, fertilization and cleavage rates Bianchi et al. submitted 2011 ≤  39 No patients ( at least one thawing cycle) Mean age (  DS)31.1  ,4   4.28 No. of thawing cycles No. of oocytes Thawed Survived (%)737 (74.2)570 (70.0)459 (70.6) Microinjected PN (%)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)

≤  39 No. of embryo transfers No. of embryos transferred No. pregnancies Pregnancy rate/embryo transfer % Pregnancy rate/thawing cycle % Pregnancy rate/patient % Multiple pregnancies (%)16 (37.2)4 (14.3)6 (31.6) No. of gestational sacs No. of FHB Implantation rate % Implantation/injected oocyte % 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 P < for all protocols in comparison to control Borini et al., unpublished

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