1. Cryopreservation
in
ART
Lt Col P R Lele
Classified Spl (Ob-Gyn)
Trained in ART
Southern Star ART Centre
CH (SC) Pune

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. 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. Nature…

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. 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. 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. 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. 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. 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

24. 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.

25. CPAs
 First successful use .. ..1949( Polge et al )
glycerol to freeze semen
 Two types
Permeating
Non Permeating

26. 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

27. 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.

28. 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

29. 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

30. 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.

31. 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)

32. 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.

33. Cryopreservation protocols
 Equilibrium Protocol /slow freezing
 Non Equilibrium Protocol /vitrification/
ultra-rapid freezing

34. Cryopreservation…
 Sperm
 Embryo
 Oocyte
 Testicular Tissue
 Ovarian Tissue

35. 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.

36. 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.

37. 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

38. 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

39. 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.

40. Sperm/Semen
 Difference in sperm cryosurvival rates
between normal semen and semen with
abnormal parameters
OTA
Cancer Pts
 Solution ….ICSI

41. How to freeze

42. 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

43. 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.

44. 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 ).

45. 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

46. 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

47. 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

48. 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

49. 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)

50. 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

51. 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

52. 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

53. 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.

54. 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

55. 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

56. Embryo Freezing
 Embryos can be frozen sucessfully by slow
and ultrarapid freezing

57. 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

58. Indication
Difficult embryo transfer
Preservation of fertility ..future offspring's
Embryo donation
Embryos for research purposes

59. When to freeze
 PN Stage
 Clevage Stage
 Blastocyst Stage

60. 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…

61. 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.

62. 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

63. "SOUTHERN STAR” ART CENTRE

64. Facilities available
Sperm Cryopreservation
Embryo cryopreservation
Oocyte Cryopreservation
Ovarian/testicular Freezing

65. Semen Preserved
No of samples frozen (2010-11)
286
No of samples thawed 118
Pregnancies with use of frozen thawed
semen samples 32

66. Embryo Freezing (2011)
No of patients 32
No of pts having frozen embryo
cycles 12
No of pregnancies 09

67. 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

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THANK YOU

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