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CLINICA VALLE GIULIA, Rome



 When and how to cryopreserve
             oocytes?
Analysis of oocyte physiology and
 molec...
www.generaroma.it



Oocyte cryopreservation:when
          and how?

    Medical reason

         Malignant diseases

   ...
www.generaroma.it



Oocyte cryopreservation : when
          and how?
  Logistic reasons

         Sperm collection probl...
www.generaroma.it



Oocyte cryopreservation: when
          and how?
  Oocyte donation program


  Oocyte banks may resul...
www.generaroma.it


Traditional freezing and/or vitrification?

Efficiency in donation program not compromised
with vitrif...
Laboratory outcomes:
Slow freezing infertile population
www.generaroma.it



Clinical application: infertile population
www.generaroma.it



                           Study design

In order to validate the effectiveness of a vitrification ap...
www.generaroma.it



                      Material & Methods

The general idea of the study was to minimize extra stress ...
www.generaroma.it



Patient population




         Rienzi et al., Human Reproduction 2010
www.generaroma.it



Laboratory outcomes

      Survival rate




      Rienzi et al., Human Reproduction 2010
Laboratory outcomes:
Vitrification egg donation program
Clinical outcomes:
Slow freezing infertile population
Clinical outcomes:
             èè+
Slow freezing infertile population
www.generaroma.it



Cumulative ongoing pregnancy rates: vitrification
www.generaroma.it



                    Study design


o The study was design as a prospective longitudinal
  cohort stud...
www.generaroma.it



                Material & Methods


o All consecutives patients undergoing ICSI treatment
 in the Ce...
www.generaroma.it



Laboratory results

                           44.6% of our patients,
                           39.9...
www.generaroma.it



Clinical results




    Ubaldi et al., Human Reproduction 2010
www.generaroma.it



           Results




                                        P=0,006




647 vitrified oocytes are ...
www.generaroma.it



      Oocyte vitrification: clinical
     application infertile population

o Embryo development is n...
www.generaroma.it



              Obstetric outcomes



Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A,
Castellón L...
Analysis of oocyte physiology and
               molecular markers
 “To him who devotes his life to science, nothing can g...
Analysis of oocyte physiology and
       molecular markers




                           membrane
                       ...
Membrane permeability

                                       Survival rates of human
                                    ...
Analysis of oocyte physiology and
       molecular markers

                        CG release and
                       ...
Cortical granules release

No evidence of cortical   Failed Fertilized
granule discharge in                          Fresh...
Zona Pellucida Hardening

                                                      900
                      Time for zona di...
Analysis of oocyte physiology and
       molecular markers

                           CG release and
                    ...
Aneuploidy and PB retention
Early reports on failure of PBII extrusion and
  increase of aneuploidy in thawed mouse oocyte...
Analysis of oocyte physiology and
       molecular markers

                              CG release and
                 ...
Meiotic spindle analysis during
           slow freezing
                           Rienzi et al., 2004




     PBS     F...
Meiotic spindle analysis during
                  slow freezing




                         50.8%
                       ...
Meiotic spindle analysis during
                 slow freezing
                                                       1.5 ...
Meiotic spindle analysis during
                    vitrification

Pre-vitrification                  Post-vitrification

...
Meiotic spindle analysis during
                                 vitrification
                                        Mei...
Analysis of oocyte physiology and
         molecular markers

                                  CG release and
Cytoplasmic...
Osmotic toxicity


                                                     Coticchio et al., 2004                          VI...
Osmotic toxicity

OOCYTE    OSMOTIC         TOLERENCE       AND      OOLEMMA
PERMEABILITY
Temperature of exposure influenc...
Analysis of oocyte physiology and
         molecular markers

                                  zona pellucida
Cytoplasmic...
Oocyte metabolism post-
             cryopreservation

METABOLISM MONITORING THROUGH PYRUVATE UPTAKE
(mouse oocytes):
Mous...
Oocyte metabolism post-
                         cryopreservation
                  5


                  4         a
Pyru...
Oocyte protein profile post-
           cryopreservation

PROTEOMIC ANALYSIS OF OOCYTE PROTEIN
PROFILES (mouse oocytes) by...
Hierarchical Clustering of Anionic Protein Profile

                            In Vivo &     Slow
                       ...
Analysis of oocyte physiology and
         molecular markers

                                       zona pellucida
Cytopl...
Possible injuries
 Oocyte aging
Oocyte safety

Oocyte cryopreservation poses certainly specific problems:

- The oolemma and not the size of MII oocyte is...
Safety of the procedures


Concerns

“The most widely emphasized concerns… are
toxicity and danger of contamination.

Unfo...
www.generaroma.it


                       CLINICA VALLE GIULIA, Roma
                     SALUS – ASI MEDICAL, Marostica
...
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When and how to cryopreserve oocytes analysis of oocyte physiology and molecular markers to improve cryopreservation methods-alpha rienzi-laura_2010

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When and how to cryopreserve oocytes- Analysis of oocyte physiology and molecular markers to improve cryopreservation methods-(By Rienzi_Laura_2010)

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When and how to cryopreserve oocytes analysis of oocyte physiology and molecular markers to improve cryopreservation methods-alpha rienzi-laura_2010

  1. 1. CLINICA VALLE GIULIA, Rome When and how to cryopreserve oocytes? Analysis of oocyte physiology and molecular markers to improve cryopreservation methods ALPHA 2010 8TH BIENNIAL CONFERENCE BUDAPEST 2010 Laura Rienzi, Rome, Italy
  2. 2. www.generaroma.it Oocyte cryopreservation:when and how? Medical reason Malignant diseases Surgical ovary removal Polycystic ovary Hyperstimulation sydrome Premature menopause etc.
  3. 3. www.generaroma.it Oocyte cryopreservation : when and how? Logistic reasons Sperm collection problem Legal reasons Restrictions in embryo cryopreservation Fate of embryos of separated couples Social reasons Wish to delay motherhood Moral reasons
  4. 4. www.generaroma.it Oocyte cryopreservation: when and how? Oocyte donation program Oocyte banks may result in - widespread availability - shortened, eliminated waiting list - safety (quarantine) - choice
  5. 5. www.generaroma.it Traditional freezing and/or vitrification? Efficiency in donation program not compromised with vitrification (Cobo et al., 2007; Nagy et al., 2007) Prospective randomized study with own oocytes no difference (Rienzi et al., 2010) The clinical pregnancy rate has doubled with the introduction of vitrification (Tulandi, 2008) Cumulative ongoing pregnancy rate with oocyte vitrification without embryo selection in a standard infertility program (Ubaldi, 2010)
  6. 6. Laboratory outcomes: Slow freezing infertile population
  7. 7. www.generaroma.it Clinical application: infertile population
  8. 8. www.generaroma.it Study design In order to validate the effectiveness of a vitrification approach for oocyte cryopreservation a prospective comparison was designed in our population of infertile patients (september 08 - march 09). This study was set-up as a non-inferiority trial with a prospective target of 240 sibling metaphase II oocytes obtained from an estimated 40 ICSI patients Oocyte fertilization rates after ICSI (per warmed oocyte and per injected oocyte) were evaluated as primary outcomes. Secondary outcomes were pronuclear morphology and embryo development Rienzi et al., Human Reproduction 2010
  9. 9. www.generaroma.it Material & Methods The general idea of the study was to minimize extra stress on oocytes often related with cryopreservation procedures, namely: 1. Long exposure to Hepes buffered media, with uncertain temperature control, for oocyte denudation and selection under the inverted microscope 2. Prolonged oocyte in vitro culture without the protection of cumulus and corona cells 3. Oocyte ageing In this way, by using randomized sibling oocytes the only difference between the fresh and the vitrified group was the vitrification procedure itself followed by 2 hours of in vitro culture. Rienzi et al., Human Reproduction 2010
  10. 10. www.generaroma.it Patient population Rienzi et al., Human Reproduction 2010
  11. 11. www.generaroma.it Laboratory outcomes Survival rate Rienzi et al., Human Reproduction 2010
  12. 12. Laboratory outcomes: Vitrification egg donation program
  13. 13. Clinical outcomes: Slow freezing infertile population
  14. 14. Clinical outcomes: èè+ Slow freezing infertile population
  15. 15. www.generaroma.it Cumulative ongoing pregnancy rates: vitrification
  16. 16. www.generaroma.it Study design o The study was design as a prospective longitudinal cohort study. o The baseline characteristics, embryological data, clinical and ongoing pregnancy rate were analyzed on a per cycle basis. o The cumulative pregnancy rate obtained with fresh and vitrified oocytes from the same stimulation cy- cle was analyzed on a per patient basis. Ubaldi et al., Human Reproduction 2010
  17. 17. www.generaroma.it Material & Methods o All consecutives patients undergoing ICSI treatment in the Centre for Reproductive Medicine GENERA between September 2nd 2008 and May 15th 2009 were considered for this study o Only patients with supernumerary oocytes available for cryopreservation were included. A single fresh attempt was included for each patient. Ubaldi et al., Human Reproduction 2010
  18. 18. www.generaroma.it Laboratory results 44.6% of our patients, 39.9% of cycles Ubaldi et al., Human Reproduction 2010
  19. 19. www.generaroma.it Clinical results Ubaldi et al., Human Reproduction 2010
  20. 20. www.generaroma.it Results P=0,006 647 vitrified oocytes are still available Ubaldi et al., Human Reproduction 2010
  21. 21. www.generaroma.it Oocyte vitrification: clinical application infertile population o Embryo development is not affected by the vitrification procedure up to day 2 o High cumulative ongoing pregnancy rates were achieved in a standard infertility program with transfers of embryos derived from fresh and subsequently vitrified eggs o Among various infertility factors, only female age influenced significantly the outcome o The overall efficiency justifies the application of this stra- tegy in routine infertility work
  22. 22. www.generaroma.it Obstetric outcomes Chian RC, Huang JY, Tan SL, Lucena E, Saa A, Rojas A, Castellón LA, García Amador MI, Montoya Sarmiento JE. Obstetric and perinatal outcome in 200 infants conceived from vitrified oocytes. Reprod Biomed online 2008 Noyes N, Porcu E, Borini A. Over 900 oocyte cryopreservation babies born with no apparent increase in congenital anomalies. Reprod Biomed online 2009
  23. 23. Analysis of oocyte physiology and molecular markers “To him who devotes his life to science, nothing can give more happiness than increasing the number of discoveries, but his cup of joy is full when the results of his studies immediately find practical applications.” Louis Pasteur Stress tollerance
  24. 24. Analysis of oocyte physiology and molecular markers membrane permeability
  25. 25. Membrane permeability Survival rates of human oocytes frozen with the same slow freezing protocol 60-65% 35-40% 75-80% (Lassalle et al., 1985) Aquaporin-9, a protein channel that Osmotic response to glycerol of can transport water and other mouse oocytes injected with solutes through the plasmalemma is Aquaporin-3 cRNA expressed in rat GV-stage but not mature oocytes (Ford et al., 2000) Aqua-3 + + Permeability Aquaporin-9 + - expression Edashige et al., 2003
  26. 26. Analysis of oocyte physiology and molecular markers CG release and ZP hardening membrane permeability
  27. 27. Cortical granules release No evidence of cortical Failed Fertilized granule discharge in Fresh cryopreserved oocytes Gook et al., 1993 Frozen Non-frozen Frozen “The immunostaining examination for CG of the frozen–thawed oocytes did not reveal evidence of the premature release of CG.” Li et al., 2005 Ghetler et al., 2006
  28. 28. Zona Pellucida Hardening 900 Time for zona dissolution (s) 800 700 600 500 400 300 200 100 0 Control Vitrified DMSO/EG (non-vitrified) Larman et al., 2006 n = greater than 60 oocytes per treatment with 3 replicates
  29. 29. Analysis of oocyte physiology and molecular markers CG release and ZP hardening membrane permeability Polar body degeneration/fusion
  30. 30. Aneuploidy and PB retention Early reports on failure of PBII extrusion and increase of aneuploidy in thawed mouse oocytes Glenister et al, 1987; Carroll et al., 1989 No. of Oocytes (%) Frozen Scored % Aneuploidy % Retention PB + 352 6.4 2.6 - 218 8.0 4.4 No increase in the rates of aneuploidy/digyny in parthenogenetically activated mouse oocytes after cryopreservation with DMSO/slow freezing Bos-Mikich and Whittingham, 1995
  31. 31. Analysis of oocyte physiology and molecular markers CG release and ZP hardening membrane permeability Meiotic spindle depolymerization Polar body degeneration/fusion
  32. 32. Meiotic spindle analysis during slow freezing Rienzi et al., 2004 PBS FS1 FS2 FREEZING THAWING TS1 TS2 TS3 PBS 3h 37°C
  33. 33. Meiotic spindle analysis during slow freezing 50.8% vs 73.1% 1.5 PrOH sucrose 0.1mol/l Coticchio et al., 2006
  34. 34. Meiotic spindle analysis during slow freezing 1.5 PrOH sucrose 0.3 mol/l 69.7% Detectable meiotic spindle (%) vs 73.1% Bianchi et al., Human Reproduction, 2005 Coticchio et al., 2006
  35. 35. Meiotic spindle analysis during vitrification Pre-vitrification Post-vitrification Post-culture 2h
  36. 36. Meiotic spindle analysis during vitrification Meiotic Spindle view and vitrification HUMAN OOCYTES Meiotic spindle intensity 2.5 2 1.5 1 0.5 0 pre-vit 0h 2h Larman, RBM on line 2007
  37. 37. Analysis of oocyte physiology and molecular markers CG release and Cytoplasmic and ZP hardening Cytoskeletron damage membrane permeability Meiotic spindle depolymerization Polar body degeneration/fusion
  38. 38. Osmotic toxicity Coticchio et al., 2004 VITRIFICATION 1 0,9 Normalised volume 0,8 Experimental 0,7 Predicted 0,6 0,5 0,4 0 60 120 180 240 300 360 420 480 540 Time (secs) SLOW FREEZING
  39. 39. Osmotic toxicity OOCYTE OSMOTIC TOLERENCE AND OOLEMMA PERMEABILITY Temperature of exposure influence shrinking (swelling) patterns - Oocyte shrinkage tolerance is about 30% of their initial volume - At 22°C, EG has a lower permeability coefficient relative to DMSO and PG - The membrane is more selective for EG and DMSO than for PG (mean reflection coefficient Sigma lower for PG) - Permeability coefficients of individual oocytes varied substantially (inherent biological variability) Van den Abbeel et al., 2007
  40. 40. Analysis of oocyte physiology and molecular markers zona pellucida Cytoplasmic and hardening Cytoskeletron damage membrane permeability Meiotic spindle depolymerization Polar body Impact on oocyte degeneration/fusion physiology
  41. 41. Oocyte metabolism post- cryopreservation METABOLISM MONITORING THROUGH PYRUVATE UPTAKE (mouse oocytes): Mouse oocytes and developing embryos following slow freezing were metabolically impaired compared with those that were vitrified …although vitrification was also associated with a decrease in nutrient utilization by the oocyte compared to controls the decrease was significantly smaller than that induced by slow freezing. Lane and Gardner, 2001; Lane et al., 2002
  42. 42. Oocyte metabolism post- cryopreservation 5 4 a Pyruvate Uptake (pmol/oocyte/h) 3 b 2 c 1 0 Control Vitrification Slow-freezing Lane and Gardner., 2001
  43. 43. Oocyte protein profile post- cryopreservation PROTEOMIC ANALYSIS OF OOCYTE PROTEIN PROFILES (mouse oocytes) by SELDI-TOF MS: Mouse oocytes following slow freezing revealed major alterations compared with those that were vitrified. Vitrified oocyctes appeared to be similar to the non-cryopreserved control oocytes... Larman et al., 2006
  44. 44. Hierarchical Clustering of Anionic Protein Profile In Vivo & Slow Vitrified Frozen Oocytes Oocytes Heat Map = Upregulated = Downregulated Larman et al., 2006
  45. 45. Analysis of oocyte physiology and molecular markers zona pellucida Cytoplasmic and hardening Cytoskeletron damage Oocyte membrane ageing permeability Meiotic spindle depolymerization Polar body Impact on oocyte degeneration/fusion physiology
  46. 46. Possible injuries Oocyte aging
  47. 47. Oocyte safety Oocyte cryopreservation poses certainly specific problems: - The oolemma and not the size of MII oocyte is the key to explain the low survival rates obtained with slow freezing. - Release of cortical granules (controversial) - Chemical toxicity from cryoprotectants (type specific) - Osmotic toxicity - Meiotic spindle depolymerization (slow freezing) - Oocyte physiology alteration (metabolism and protein profile) especially true for slow freezing
  48. 48. Safety of the procedures Concerns “The most widely emphasized concerns… are toxicity and danger of contamination. Unfortunately, available vitrification methods still struggle with these problems to date” Son and Tan, 2009
  49. 49. www.generaroma.it CLINICA VALLE GIULIA, Roma SALUS – ASI MEDICAL, Marostica Ginecologia: Embriologia: Filippo Ubaldi Laura Rienzi Elena Baroni Stefania Romano Silvia Colamaria Laura Albricci Maddalena Giuliani Antonio Capalbo Fabio Sapienza Roberta Maggiulli Matteo Buccheri Benedetta Iussig Nicoletta Barnocchi

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