2. Why Preservation….
The basic function of any species is to
conserve … preserve…. Procreate
Homo sapeins have taken clues from nature
to further improve…
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.
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
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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.
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
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
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
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
