Cryopreservation in Assisted Reproduction


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Cryopreservation in Assisted Reproduction

  1. 1. Cryopreservation in ART Lt Col P R Lele Classified Spl (Ob-Gyn) Trained in ART Southern Star ART Centre CH (SC) Pune
  2. 2. Why Preservation….  The basic function of any species is to conserve … preserve…. Procreate  Homo sapeins have taken clues from nature to further improve…
  3. 3. Cryopreservation— …cryobiology Preserves the normal function of cells or tissues by a reduction in temperature below which normal chemical reactions do not take place.
  4. 4. Nature…
  5. 5. ORIGINS OF CRYOBIOLOGY Could be traced down to ancient Egyptians “New Experiments and Observations Touching Cold”(London, 1683) by Sir Robert Boyle-first scientific account of this science . Cryopreservation of reproductive cells dates as early as 1776, with the report by Spallazani of sperm freezing
  6. 6. 1900…developments Cryogenic equipment closed systems based on liquid nitrogen Joule–Thomson cooling with mixed gases, etc. Monitoring techniques Extension of the list of diseases that have been successfully treated by cryomedicine 1964 of two major scientific societies in this field  The Society for Cryobiology The Society for Low Temperature Biology.
  7. 7. Cryopreservation— …cryobiology The first live births following frozen- thawed embryo transfer were reported in 1984 and 1985 by groups in Australia, the Netherlands and the UK.
  8. 8. In Cryopreservation Major classes of physical stresses that cells are exposed to during freezing: 1. Direct effects of reduced temperature 2. Physical changes associated with ice formation .
  9. 9. Direct effects of reduced temperature  Cold shock injury  damage to cell structure and function arising from a sudden reduction in temperature  Modifications in membrane permeability and changes in the cytoskeletal structure  species-specific ..not evident …. human sperm and embryos.  freezing oocytes, ovarian tissue and testicular tissue, the possibility of sublethal injuries does exist…….breakdown of mitotic spindles.
  10. 10. Physical Changes associated with ice formation… Supercooling Cool below their melting point before nucleation of ice Temp of water could be brought below -40 in controlled environment before ice formation This depends on Temperature / rate of cooling / vol / exclusion of atm ice / purity of particles Seeding introduction of a crystal to an undercooled solution. “Nucleation” is the initiation of ice other than by seeding
  11. 11. CRYOPROTECTANTS  Cryoprotectant additives (CPAs) Simple, low molecular weight molecules with high water solubility and low toxicity Protect cells by stabilizing intracellular proteins Reducing or eliminating lethal intracellular ice formation, Moderating the impact of concentrated intra- and extracellular electrolytes.
  12. 12. CPAs  First successful use .. ..1949( Polge et al ) glycerol to freeze semen  Two types Permeating Non Permeating
  13. 13. Permeating CPAs glycerol, ethylene glycol (EG), 1,2- propanediol (PROH) and dimethylsulfoxide (DMSO) penetrate the cell membrane more slowly than water stabilize intracellular proteins, reduce the temperature at which intracellular ice forms
  14. 14. Permeating CPAs minimize osmotic damage due to electrolyte concentration effects. Gly penetrates tissue less readily than DMSO, PROH and EG. These newer cryoprotectants have higher water solubility, rapid tissue penetration and induce less osmotic damage at high concentrations.
  15. 15. Non Permeating CPAs Sugars, polymers and amphipathic compounds Raffinose and lactose decrease the percentage of unfrozen water and/ or decrease salt concentrations. Glycine, proline and trehalose are amphipathic compounds that interact with membrane lipids and proteins to alter phase transitions and hydration status
  16. 16. CPAs  CPAs should always be used in combination with non-permeating osmolytes such as sucrose or mannitol.  These act as osmotic buffers to protect against cell swelling during the addition/removal of CPAs
  17. 17. SIDE EFFFECTS-CPA  True chemical toxicity (Baxter and Lathe, 1971; Fahy, 1986).  In vitrification methods very high concentrations of these compounds are necessary to achieve and maintain a vitreous state.  The precise nature of the toxic effects of CPA remains, to a large degree, uncertain.
  18. 18. SIDE EFFECTS - CPA  CPA have been shown to alter cytoskeletal components in mammalian oocytes, particularly the filamentous actin network and meiotic spindle Johnson and Pickering, 1987;Vincent et al., 1990;Vincent and Johnson, 1992)
  19. 19. BASICS OF CRYOPRESERVATION  Incubating the cells in solutions in which CPA compounds are dissolved.  Then cells are cooled to a low sub-zero temperature  Specimens are typically held at the temperature of liquid nitrogen; -196 °C.  At the appropriate time, the specimen is warmed, washed free of the CPA, and used in whatever manner is deemed appropriate.
  20. 20. Cryopreservation protocols  Equilibrium Protocol /slow freezing  Non Equilibrium Protocol /vitrification/ ultra-rapid freezing
  21. 21. Cryopreservation…  Sperm  Embryo  Oocyte  Testicular Tissue  Ovarian Tissue
  22. 22. Sperm/Semen  Used successfully for artificial insemination (AI), intrauterine insemination (IUI) and IVF.  Although freeze-thawing does produce damage to the cells with loss of up to 50% of pre-freeze motility, since large numbers of cells are available, successful fertilization can be achieved even with low cryosurvival rates.
  23. 23. Indications  Men who can not produce semen sample on demand at the time of IUI or IVF.  Men who cannot be present at IUI or IVF time.  Person who has variation in sperm count.  Cancer pts undergoing chemo or radiotherapy which might lead him to sterility.
  24. 24. Indications  Before vasectomy  Cases prone to azoospermia  As a part of semen banking  In cases with severe male factor infertility  In couples where the partner is a frequent traveler
  25. 25. Effects of cryopreservation on Sperm  Sperm membranes have an unusual lipid composition,  Reduction in temperature alters the membrane lipid organization and modifies the kinetics or intra-membrane proteins, leading to lowered permeability and loss of fluidity.  Loss of fluidity…poor sperm survival
  26. 26. Effects of cryopreservation on Sperm  Frozen/thawed sperm behave in a similar way to capacitated sperm, which may lead to a shortened lifespan within the female tract; therefore the timing of insemination is important when using frozen sperm samples.
  27. 27. Sperm/Semen  Difference in sperm cryosurvival rates between normal semen and semen with abnormal parameters OTA Cancer Pts  Solution ….ICSI
  28. 28. How to freeze
  29. 29. Methods to improve  Sperm preparation in order to remove immotile and damaged sperm prior to freezing may help to select a population of sperm with a better chance of survival.  Use of stimulants such as pentoxifylline may also improve survival after thawing
  30. 30. Cryopreservation of testicular and epididymal sperm  Advantage…..sperm and oocyte retrieval procedures may be carried out on separate occasions  Generally epididymal and testicular samples are cryopreserved using same protocols developed for ejaculated sperm this may not be optimal.
  31. 31. Cryopreservation of testicular and epididymal sperm  Water permeability of testicular sperm, a major factor in determining the cellular response to freezing, is very different from that of ejaculated sperm  Very few sperm, sperm can be frozen individually or in small groups by injecting them into empty zona pellucida “shells,” (Walmsley et al ., 1999 ).
  32. 32. Testicular Biopsy samples  Freezing whole biopsy samples without prior processing is not recommended, as CPAswill not equilibrate evenly throughout the tissue.  Pieces of macerated or minced tissue can…frozen with some success.  Testicular sperm retain their motility on thawing if they have been incubated 24–48 hours before freezing  Doubt about sperm viability after thawing….HOS
  33. 33. Cryop.. semen for cancer pts  Young and adolecent ….progress in oncology have improved survival  But in 6 – 7 wks following t/t majority would be azoospermic  Currently no pretretment parameters for return to fertiltiy  three sperm samples are collected before chemotherapy is initiated
  34. 34. Cryop.. semen for cancer pts  Prior to sample collection for storage Screened for hepatitis B and C and HIV.  In future….. autotransplantation of cryopreserved testicular tissue may become an alternative option
  35. 35. Oocyte Cryopreservation  Admi aur Ghoda kabhi buddha nahi hota hain….  Female steadily but surely loses her eggs from birth to menopause.  Preservation of oocytes by vitrification will provide the ''aging'' woman who has had to delay … pregnancy
  36. 36. Oocyte Cryopreservation  Human oocytes are particularly susceptible to freeze-thaw damage due to their size and complexity.  They must not only survive thawing, but also preserve their potential for fertilization and development.  First pregnancies …in the 1980s (Chen, 1986 ; Al Hasani et al ., 1987)
  37. 37. Oocyte Cryopreservation Indication Prior to cancer treatments like chemo/radiotherapy Prior to organ transplant therapy (liver transplant, bone marrow transplant, etc.;) In severe Poly Cystic Ovarian Syndrome cases Single women wanting to preserve the fertility (Social Egg Freezing) In cases where male partner fails to produce semen sample on the day of IVF after egg pickup and ICSI/IVF needs to be posponed
  38. 38. Oocyte  Pre requisite HIV, Hepatitis B&C and VDRL The women needs to undergo COH Eggs will be collected under general anesthesia and frozen with VITRIFICATION
  39. 39. Ovarian tissue cryopreservation  Cryopreservation and banking of ovarian tissue ..another strategy for fertility conservation  Option is best suited for patients who are less than 30 years old and have had no previous chemo- or radiotherapy.  The uterus must be functional and the patient should have a high probability of long-term survival after treatment
  40. 40. Ovarian tissue cryopreservation Advantages of freezing ovarian tissue rather than oocytes: Small pieces of tissue contain very large numbers of primordial follicles and can be stored for children as well as for young adults. Laparoscopic ovarian biopsy/oophorectomy can be carried out rapidly before chemotherapy, any time during the menstrual cycle, thereby avoiding delays in initiating therapy.
  41. 41. Ovarian tissue cryopreservation Advantages of freezing ovarian tissue rather than oocytes: Germline cells are removed from cytotoxic harm and the entire tissue can be returned to the patient by grafting. Storing cortical tissue theoretically preserves natural cell–cell interactions and intra-ovarian signals, and graft ing can potentially restore both steroidogenic and gametogenic function – both important factors for the quality of life of the patient
  42. 42. Ovarian tissue …..  Fragments or thin slices of ovarian cortex  Whole or hemi ovaries  Dissected intact isolated primordial follicles and denuded primordial oocytes from isolated primordial follicles
  43. 43. Embryo Freezing  Embryos can be frozen sucessfully by slow and ultrarapid freezing
  44. 44. Indication Supernumery embryos following fresh embryo transfer. In case of ovarian hyperstimulation syndrome Jeopardizing implantation apparent. Thin endometrium Thick endometrium Fluid in endometrial cavity Bleeding in cycle Polyp
  45. 45. Indication Difficult embryo transfer Preservation of fertility ..future offspring's Embryo donation Embryos for research purposes
  46. 46. When to freeze  PN Stage  Clevage Stage  Blastocyst Stage
  47. 47. PN Stage  Timing of pronucleate freezing is crucial, and the process must be initiated while the pronuclei are still distinctly apparent, no later than 20–22 hours after insemination  If done later…
  48. 48. Cleavage Stage  2 to 8 cell embryos should be of good quality, grade 1 or 2, with less than 20% cytoplasmic fragments.  Uneven blastomeres and a high degree of fragmentation jeopardize survival potential; embryos with damage after thawing may still be viable and result in pregnancies, but their prognosis for implantation is reduced.
  49. 49. Became routine after media for effective extended culture became available during the 1990s Assisted hatching and cryopreservation could be offered to obviate the problems zona hardening Better pregnancy rates in surviving embryos Blastocyst
  51. 51. Facilities available Sperm Cryopreservation Embryo cryopreservation Oocyte Cryopreservation Ovarian/testicular Freezing
  52. 52. Semen Preserved No of samples frozen (2010-11) 286 No of samples thawed 118 Pregnancies with use of frozen thawed semen samples 32
  53. 53. Embryo Freezing (2011) No of patients 32 No of pts having frozen embryo cycles 12 No of pregnancies 09
  54. 54. Conclude…. The current focus in ART to improve techniques for better success rates, decrease the treatment cost and at the same time preserve the fertility potential of young individuals. Cryopreservation of gametes and embryos is one such initiative . The same is available in your hospital