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Dr G A RAMA RAJU
Embryo
                                sperm              Oocyte

                Key issues
                      in
                                                            Ovary
                vitrification                               /testis


   Slow freezing
   /vitrification                                                     Safety



Components
                                        Over view                       New
    of
cryobiology                                                             born

    .
Nature
?
? A vitrified frog felt as a rock
Cell injury.




   1 Cell size shape ,permeability ,individual quality
     and sensitivity.
   2 cryoprotectant toxicity
   3 cooling and warming.
The land mark publication
       “Slow cooling”
           Trounson and Mohr
                          1983




       A long gap…………………………………….
PLANER COMPUTERISED FREEZER
Father Basile.j.luyet

work between 1937-1958



   Innovator :




                           1952 Time magazine
Early adopter: PARKES




                        15
Publication from different countries
First Concept     • Add cryoprotectant


Second Concept     • Cool cells to -196 & store


Third Concept      • Warm/Thaw


 Fourth concept.   • Remove cryoprotectant
Results
Time             Slow
Cost
               freezing
vitrificatio
n
Variables Variable that effect vitrification
of vitrification that can profoundly influence its effectiveness




                                                                   19
Biology of Reproduction December 1, 2002 vol. 67 no. 6 1671-1680
                                                                   20
Over view of current
work.
OSMOTIC ALTERATION
                                      OTHER SACCHARIDES
     Sucrose
                     CPA
                                      DMSO
 Ethylene glycol                      PROPYLENE GLYCOL
                                      GLYCEROL


Gamete media g1
                                    HEPES BUFFERED MEDIA
Composition of Vitrification Solutions

                                                                                           V1:
             ES: 7.5%                                                                    BUFFER
              of EG
             + 7.5% of
               DMSO
                                                                                   V2: EG + Propanediol


   VS: 15% of EG + 15% of DMSO +
         0.6M of SUCROSE
                                                                               V3: EG + Propanediol + FICOLL



Sage Bio Pharma                                                                       Vitrolife


                                              Rama Raju et al

                                                         ES: 7.5%
                                                           of EG
                                                       + 7.5% of 1,2
                                                       Propanediol



                                                 VS: 15% of EG + 15% of 1,2
                                               Propanediol + 0.5M of SUCROSE
Composition of Warming Solutions


                                                           W1:
                                                          Sucrose
     1M Sucrose


                                                         W2: Sucrose



     0.5M Sucrose
                                                         W3: Sucrose



Sage Bio Pharma                                        Vitrolife

                          Rama Raju et al
                               1M Sucrose




                              0.5M Sucrose




                              0.25M Sucrose



                                  Medi Cult
   Vitrification and slow freezing
    similar results
Japanese group




      vajata


  We are working on
removal of water from
oocyte and working on
         that
33
The odds of post-thawing survival rate of cleavage-stage embryos after vitrification or slow freezing




                    Devroey, P. et al. Hum Reprod Update 2009 15:391-408; doi:10.1093/humupd/dmp012




Copyright restrictions may apply.
Blastocyst
38
39
Ultraviolet light to sterilize the liquid nitrogen
Use of hospital       Long            Increase risk of
                                                           epilepsy
   services       hospitalization        infection



                       Sleep                              Behavior
Cerebral palsy                         Convulsions
                   disturbances                           problems



                             Congenital
                            malformation


                                    Need for long tern follow up to look deep
Need for long tern follow up to look deep
Neonatal outcome of day 3
        embryos
   Hans Tinneberg my mentor
Visakhapatnam Beach .   summary

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Vitrification in IVF

  • 1. Dr G A RAMA RAJU
  • 2. Embryo sperm Oocyte Key issues in Ovary vitrification /testis Slow freezing /vitrification Safety Components Over view New of cryobiology born .
  • 3.
  • 5. ?
  • 6. ? A vitrified frog felt as a rock
  • 7.
  • 8. Cell injury. 1 Cell size shape ,permeability ,individual quality and sensitivity. 2 cryoprotectant toxicity 3 cooling and warming.
  • 9. The land mark publication “Slow cooling” Trounson and Mohr 1983 A long gap…………………………………….
  • 11.
  • 12.
  • 13.
  • 14. Father Basile.j.luyet work between 1937-1958 Innovator : 1952 Time magazine
  • 17. First Concept • Add cryoprotectant Second Concept • Cool cells to -196 & store Third Concept • Warm/Thaw Fourth concept. • Remove cryoprotectant
  • 18. Results Time Slow Cost freezing vitrificatio n
  • 19. Variables Variable that effect vitrification of vitrification that can profoundly influence its effectiveness 19
  • 20. Biology of Reproduction December 1, 2002 vol. 67 no. 6 1671-1680 20
  • 21. Over view of current work.
  • 22. OSMOTIC ALTERATION OTHER SACCHARIDES Sucrose CPA DMSO Ethylene glycol PROPYLENE GLYCOL GLYCEROL Gamete media g1 HEPES BUFFERED MEDIA
  • 23.
  • 24. Composition of Vitrification Solutions V1: ES: 7.5% BUFFER of EG + 7.5% of DMSO V2: EG + Propanediol VS: 15% of EG + 15% of DMSO + 0.6M of SUCROSE V3: EG + Propanediol + FICOLL Sage Bio Pharma Vitrolife Rama Raju et al ES: 7.5% of EG + 7.5% of 1,2 Propanediol VS: 15% of EG + 15% of 1,2 Propanediol + 0.5M of SUCROSE
  • 25. Composition of Warming Solutions W1: Sucrose 1M Sucrose W2: Sucrose 0.5M Sucrose W3: Sucrose Sage Bio Pharma Vitrolife Rama Raju et al 1M Sucrose 0.5M Sucrose 0.25M Sucrose Medi Cult
  • 26.
  • 27.
  • 28.
  • 29. Vitrification and slow freezing similar results
  • 30.
  • 31. Japanese group vajata We are working on removal of water from oocyte and working on that
  • 32.
  • 33. 33
  • 34. The odds of post-thawing survival rate of cleavage-stage embryos after vitrification or slow freezing Devroey, P. et al. Hum Reprod Update 2009 15:391-408; doi:10.1093/humupd/dmp012 Copyright restrictions may apply.
  • 35.
  • 36.
  • 38. 38
  • 39. 39
  • 40. Ultraviolet light to sterilize the liquid nitrogen
  • 41. Use of hospital Long Increase risk of epilepsy services hospitalization infection Sleep Behavior Cerebral palsy Convulsions disturbances problems Congenital malformation Need for long tern follow up to look deep
  • 42.
  • 43. Need for long tern follow up to look deep
  • 44.
  • 45. Neonatal outcome of day 3 embryos
  • 46.
  • 47. Hans Tinneberg my mentor

Editor's Notes

  1. Falling pictures(Intermediate)To reproduce the picture effects on this slide, do the following:On the Home tab, in the Slides group, click Layout, and then click Blank.On the Insert tab, in the Illustrations group, click Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the Picture Styles group, click More, and then click Beveled Matte, White (first row, second option from the left). Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, under Size and rotate, in the Rotation box, enter 5°.Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatPicture dialog box launcher. In the FormatPicture dialog box, in the left pane, click Shadow. In the Shadow pane, click the button next to Presets, and then under Outer click Offset Diagonal Bottom Left (first row, third option from the left). In the Distance box, enter 20 pt. Drag the picture into the top right corner of the slide.Select the picture. On the Home tab, in the Clipboard group, click the arrow under Paste, and then click Duplicate.Drag the second (duplicate) picture to the left middle of the slide.Select the second picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, under Size and rotation, in the Rotation box, enter 0°.Right-click the second picture and click Change Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the second picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatPicture dialog box launcher. In the FormatPicture dialog box, in the left pane, click 3-D Rotation. In the 3-D Rotation pane, click the button next to Presets, under Perspective click Perspective Relaxed (second row, third option from the left), and then do the following: In the X box, enter 0°.In the Y box, enter 320°.In the Z box, enter 10°.In the Perspective box, enter 80°. On the Home tab, in the Drawing group, click Arrange, and then click Send to Back. Select the second picture. On the Home tab, in the Clipboard group, click the arrow under Paste, and then click Duplicate.Drag the third picture to the bottom right corner of the slide, under the first picture.Right-click the third picture and click Change Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the third picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatShape dialog box launcher. In the FormatPicture dialog box, in the left pane, click 3-D Rotation. In the 3-D Rotation pane, click the button next to Presets, under Perspective click Perspective Relaxed (second row, third option from the left), and then do the following: In the X box, enter 0°.In the Y box, enter 300°.In the Z box, enter 0°.In the Perspective box, enter 60°. On the Home tab, in the Drawing group, click Arrange, and then click Send to Back. To reproduce the background effects on this slide, do the following:Right-click the slide background area, and then click Format Background. In the Format Background dialog box, click Fill in the left pane, select Gradient fill in the Fill pane, and then do the following:In the Type list, select Linear.Click the button next to Direction, and then click Linear Down (first row, second option from the left). Under Gradient stops, click Add or Remove until two stops appear in the drop-down list.Also under Gradient stops, customize the gradient stops that you added as follows:Select Stop 1 from the list, and then do the following:In the Stop position box, enter 70%.Click the button next to Color, and then click Black, Text 1, Lighter 5% (sixth row, second option from the left). Select Stop 2 from the list, and then do the following: In the Stop position box, enter 100%.Click the button next to Color, click More Colors, and then in the Colors dialog box, on the Custom tab, enter values for Red: 87, Green: 78, Blue: 51. 
  2. Falling pictures(Intermediate)To reproduce the picture effects on this slide, do the following:On the Home tab, in the Slides group, click Layout, and then click Blank.On the Insert tab, in the Illustrations group, click Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the Picture Styles group, click More, and then click Beveled Matte, White (first row, second option from the left). Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, under Size and rotate, in the Rotation box, enter 5°.Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatPicture dialog box launcher. In the FormatPicture dialog box, in the left pane, click Shadow. In the Shadow pane, click the button next to Presets, and then under Outer click Offset Diagonal Bottom Left (first row, third option from the left). In the Distance box, enter 20 pt. Drag the picture into the top right corner of the slide.Select the picture. On the Home tab, in the Clipboard group, click the arrow under Paste, and then click Duplicate.Drag the second (duplicate) picture to the left middle of the slide.Select the second picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, under Size and rotation, in the Rotation box, enter 0°.Right-click the second picture and click Change Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the second picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatPicture dialog box launcher. In the FormatPicture dialog box, in the left pane, click 3-D Rotation. In the 3-D Rotation pane, click the button next to Presets, under Perspective click Perspective Relaxed (second row, third option from the left), and then do the following: In the X box, enter 0°.In the Y box, enter 320°.In the Z box, enter 10°.In the Perspective box, enter 80°. On the Home tab, in the Drawing group, click Arrange, and then click Send to Back. Select the second picture. On the Home tab, in the Clipboard group, click the arrow under Paste, and then click Duplicate.Drag the third picture to the bottom right corner of the slide, under the first picture.Right-click the third picture and click Change Picture. In the Insert Picture dialog box, select a picture, and then click Insert. Select the third picture. Under Picture Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Size and Position dialog box, on the Size tab, resize or crop the picture as needed so that under Size and rotate, the Height box is set to 2.93” and the Width box is set to 4.41”. Resize the picture under Size and rotate by entering values into the Height and Width boxes. Crop the picture under Crop from by entering values into the Left, Right, Top, and Bottom boxes. Under Picture Tools, on the Format tab, in the bottom right corner of the Picture Styles group, click the FormatShape dialog box launcher. In the FormatPicture dialog box, in the left pane, click 3-D Rotation. In the 3-D Rotation pane, click the button next to Presets, under Perspective click Perspective Relaxed (second row, third option from the left), and then do the following: In the X box, enter 0°.In the Y box, enter 300°.In the Z box, enter 0°.In the Perspective box, enter 60°. On the Home tab, in the Drawing group, click Arrange, and then click Send to Back. To reproduce the background effects on this slide, do the following:Right-click the slide background area, and then click Format Background. In the Format Background dialog box, click Fill in the left pane, select Gradient fill in the Fill pane, and then do the following:In the Type list, select Linear.Click the button next to Direction, and then click Linear Down (first row, second option from the left). Under Gradient stops, click Add or Remove until two stops appear in the drop-down list.Also under Gradient stops, customize the gradient stops that you added as follows:Select Stop 1 from the list, and then do the following:In the Stop position box, enter 70%.Click the button next to Color, and then click Black, Text 1, Lighter 5% (sixth row, second option from the left). Select Stop 2 from the list, and then do the following: In the Stop position box, enter 100%.Click the button next to Color, click More Colors, and then in the Colors dialog box, on the Custom tab, enter values for Red: 87, Green: 78, Blue: 51.