Frozen Life Pankaj Talwar
INDEX
A
Absorbed dose 162
Actual cooling curve 125
Air space inside straws 122
Alcohol bath 126
Assisted reproductive technologies
freezing 198, 216
embryo selection 198
general guidelines 198
media preparation 198
method 200
ampule loading 200
freezing program 200
media handling 200
straw loading 200
thawing 200
Auto-fill system 131, 132
Automatic seeding 128
B
Biocontainment 134
Blastocyst freezing 514
blastocyst freezing media 515
blastocyst freezing simplified 515
granding of embryos 514
why freeze embryos at the
blastocyst stage 514
Blastocyst thawing 521
blastocyst thawing simplified 522
evaluation of thawing 521
injuries during blastocyst
thawing 521
transfer of frozen-thawed
embryo 521
Blastocyst vitrification 180
artificial shrinkage 183
clinical results 184
artificial shrinkage
procedures 185
perinatal outcome 186
vitrified blastocysts transfer 184
grading of blastocysts 182
patients, embryo culture 182
troubleshooting 181
warming of blastocysts 181
C
Catastrophic failure 131
CBS high security straws 103
CBS high security vitrification kit 112
Choline substitution 39
Closed-pulled straw 273
Controlled rate freezing 94
Conventional freezing of embryos 296
cryoprotectant additives 297
cryoprotective agents 297
detrimental effect 300
embryo cryopreservation protocols 301
blastocyst freezing method 301
freezing of pre-implantation stage embryo 301
ethical considerations 302
evaluation of embryo survival 300
freezing at different developmental stages 300
principle of cryopreservation 297
safety of the procedure 302
steps involved 297
cooling rate 299
embryo loading 298
embryo thawing 299
equilibration 298
seeding 298
storage 299
washing and dilution of cryoprotectant 300
Conventional slow cooling 139
Cooling 124
rates 94
Cooling and warming 91
rates 95
Cord blood banking 402, 418
advantages 402
collection of blood 418
components 403
accreditation agencies 414
accreditation of cellular therapy 414
American Association of Blood Bank 414
biosafety cabinet 405
CD34 and its relevance 412
cell based assays 409
controlled rate freezer and storage 406
cord blood collection process 404
counseling and informed consenting 403
cryopreservation 408
donor screening 404
effects of CB collection on the neonate 404
engraftment 412
equipments required 405
existing limitations of usage of UCB 413
factors that could affect UCB engraftment 412
forming cell (CFU) assays 409
graft assessment by colony 409
graft versus host disease 411
HLA antigens 409
HLA compatibility 411
HLA match search process 411
human leukocyte antigen tests 409
infectious diseases testing 408
infrastructure 405
labeling 408
liquid nitrogen storage system 406
manpower 407
methods used for DNA-based typing 410
microbial cultures 408
platelet engraftment 412
post-thaw removal 411
post-thaw washing 411
processing 407
regulatory perspectives 413
role of HLA matching 411
setting up of collection centers 403
success cord blood transplant 413
testing of cord blood units 408
transportation to transplant centers 411
cryopreservation 420
automated devices for processing 422
containers for storage 422
controlled rate freezing 421
cryoprotectants 420
freezing rate 421
principle 420
release cord blood 422
testing 422
thawing 422
washing step 423
donor recruitment 418
future trends 423
processing 419
setting up of a cord blood bank 403
types of banking
autologous banking 402
family banking 402
types of transplantation
related UCB
transplantation 403
unrelated UCB transplantation 403
Cryobanking 88, 132
brief history 88
overview 88
Cryobiology 90, 139, 453
conventional freezing methods 141
CPA-free sperm
cryopreservation 142
cryoinjury
macromolecules 140
sugars 140
cryoperservation of human ovarian
tissue 143
cytoskeletal stabilizer 142
dehydration of embryo and
blastocyst 142
immature human oocyte
cryopreservation 143
principles 139
conventional slow cooling 139
vitrification 140
recent advances 142
Cryocans 5
Cryogenics 453
Cryoinjury 140
Cryoloop 273
Cryonics 453
Cryopreservation 32, 453
cryoprotectants 32, 34
mechanism of action 33
nonpermeating 35
permeating 34
early clinical experience 37
disruption of the meiotic
spindle 37
zona pellucida hardening 38
methods of freezing
rapid freeze (vitrification) 36
slow freeze method 36
outcomes 40
recent developments 38
choline substitution 39
slow freeze method 38
trehalose 39
vitrification 39
stages 33
Cryopreservation of human
spermatozoa 217
Cryopreservation of semen
contaminated with hepatitis C
virus 284
background 284
materials and methods 285
design of the study 285
detection of HVC RNA 285
human protamine-2 gene
amplification 285
influence of the
cryoprotectant 285
seminal samples 285
results 285
analysis of the rinsing
water 285
influence of the cryoprotectant
on 285
material controls 286
Cryoprotectants 32, 34, 118, 194, 455
Cryoprotective agents 93, 94, 327
Cryosurvival 129
Cryotanks 132, 136
Cryotip 273
D
Damage caused by freezing 91
Dehydration threshold 90
Devitrification of embryo 510
principles
quick warming 510
warming using cryoloop technique 512
Disinfection 123
Divided storage 133
Dose equivalent 163
E
Egg freezing 253
Electron microscopic grids 272, 508
Embryo freezing 192, 252, 477
disadvantages 193
embryo characteristics 195
embryo freezing simplified 478
cryoscan TM 486
fertisafe 485
particle counting 485
pH meter 484
thermodisk 486
VF hygiene monitoring 485
volatile organic compound monitoring 484
equipments for freezing 195
ethical presentation 202
historical background 192
limitations 194
materials required 478
need for cryopreservation 193
principles 194
protocols and methods 196
blastocyst freezing 198
cryopreservation of
prezygotes 198
principles of cryofreezing of human embryos 477
selection of embryos 478
Embryo freezing laboratory 49
Embryo thawing
cryoprotectants used 488
principles 487
thawing simplified 488
time of thawing and endometrial
preparation 488
Embryo vitrification 176, 502
alternative unique vitrification
device 508
principles of vitrification 502
protocols and clinical results 176
vitrification using crytop 178
vitrification using conventional cryostraws 176
vitrification using cryoloops 177, 505
troubleshooting 179
conventional vitrification 179
ultrarapid vitrification cryotop 180
ultrarapid vitrification:
cryoloop 179
vitrification simplified 503
Embryogenesis 360, 369
Embryonic development in vitro 260
Embryonic stem cells 391
cryopreservation 393
conventional slow freezing 395
programmable slow freezing 394
vitrification 396
evidence supporting the
potential 392
surface antigen markers 392
Embryos cryopreservation 41, 194
Embryos thawing 194
Equipment quality control 54
centrifuges 54
cryopreservation equipment 55
dry shippers/transport units 55
laboratory environmental control 56
laminar flow biohazard hood 54
Eutectics 160
F
Fertility preservation in cancer
patients 373
cancer treatment affect fertility 373
counseling 382
effects of chemotherapy on adult men
characteristics of gonadal toxicity 374
combination regimens 376
individual drugs 375
radiation therapy 376
effects of cytotoxic agents on adult women
characteristics of gonadal 377
combination regimens 378
individual drugs 377
radiation therapy 378
effects of cytotoxic agents on children
biologic considerations in boys 378
biologic considerations in girls 379
extent of gonadal toxicity in boys 378
extent of gonadal toxicity in girls 379
genetic concerns
biologic considerations 381
pregnancy outcome 382
gonadal dysfunction after cranial irradiation 379
preservation of fertility, hormone levels and sexual function
assisted reproductive technologies 380
choice of regimens 380
gonadal shielding 380
optimization of fertility after treatment 380
pharmacologic attempts at preserving fertility 381
requirements for pregnancy 373
Fertilization rates 259
Freezing point 160
Frozen embryo transfer 359
advantages 360
clinical aspects 359
factors influencing the embryogenesis 360
freezing program 364
frozen embryo transfer 365
thawing 364
indications 359
laboratory aspects 361
optimizing embryo transfer 362
single frozen embryo transfer 363
technique of embryo transfer 362
protocol at Southened fertility 363
cryo-solutions 363
embryo selection 363
freezing 363
protocols for endometrial preparation 360
G
Gas micropipettes 272
Gas sensing monitor 8
Gel-loading tips 273
Glass 160
Glass transition temperature 130, 156
Gryoprotectants 31
DMSO 35
glycerol 35
sucrose 35
H
Hematopoietic stem cell transplantation
cryopreservtives 424
durability 426
freezing and freezing rate 425
thawing and washing procedure 427
Hematopoitic progenitor cell 417
Hemi straw system 273
Hen’s egg yolk 120
High security straws 122
Human oocyte cryopreservation 23
effects 24
various cellular organelles
microtubules 23
nuclei and nuclear envelope 25
oocyte cytoskeleton 23
zona pellucida 25
Human sperm cryobanking 325
applications
donor semen cryopreservation 326
fecundity of cryopreserved donor semen 326
semen autoconservation 326
historical perspective 325
issues 327
cooling procedures 334
cryoprotectants 327
packaging 329
to seed or not to seed 335
storage 335
at what temperature 335
auto-fill systems and alarms 340
cryogenic storage tanks 340
divided storage 340
liquid or vapor phase storage 338
monitoring cryogenic storage tank 340
post-thaw processing 340
reducing risk 339
risk of cross-contamination 336
sterilizing the controlled rate freezer 339
I
Infertility Treatment Act 1995 434
Intracytoplasmic sperm injection 313
adding sperm sample to the injection dish 317
adding sperm to injection dish 317
preparation of injection dish 317
selection of viable immotile sperm by HOS 317
cryopreservation 314
adding cryoprotectant 315
cryoprotectant 314
freezing 315
maintenance of sperm bank 316
MESA samples 315
post-thaw testing 316
preparation of sperm samples 314
TESE samples 315
disposal of frozen sperm 318
sperm preparation on the day
of ICSI 316
surgical retrieval of sperm 313
Ionising radiation 162
dose consideration in female patients 165
before treatment 167
general considerations 167
maintenance of fertility 166
precautions 167
effects of radiation 163
female fertility 164
male fertility 164
follow-up after radiation
therapy 169
measurement
absorbed dose 162
dose equivalent 163
organ dose 163
radiation exposure 162
options 168
post-chemo-radiotherapy treatment
egg storage 168
embryo storage 168
hormonal manipulation 168
hormone replacement therapy 168
ovarian cryopreservation 168
ovarian transposition 168
Isothermal vapor storage 131
IUI-ready sperm samples 119
L
Laparoscopy in ovarian tissues cryopreservation
effects of radiation on ovarian
function 385
ovarian tissue
cryopreservation 388
ovarian transplantation 387
surgical techniques for ovarian
resection 388
preservation of ovarian function
ovarian transposition 386
techniques for ovarian
transposition 386
Latent heat 19, 454
heat of fusion 127
plateau 147
Lethal zone 124
Liquid nitrogen 3
application 7
containers 5
first aid 9
hazards 7
identification 4
production 5
properties 4
cold boiling point 4
Leidenfrost effect 4
physical and chemical 4
smoking 4
safety measures while handling 6
Low temperature phenomena 92
M
Mechanical freezer 130
Meiotic spindle 37, 141
Melting point 156
Microbial and viral pathogens 289
choice of liquid nitrogen
refrigerator 292
choice of sample container 291
cryopreservation procedure 291
materials and methods 289
viability of microorganisms and viruses 289
Nucleation 156
O
Oocyte bank 144
Oocyte cryobiology 22
Oocyte cryopreservation 37, 40, 204
advancements in the field 205
clinical outcome and case
reports 207
historical overview 204
indication 205
limitation 207
techniques
oocyte freezing protocol 205
oocyte vitrification protocols 206
Oocyte freezing 492
current methodologies
slow freezing 492
ultra-rapid freezing (vitrification) 493
oocyte freezing simplified 493
principles of oocyte freezing 492
Oocyte thawing 499
oocyte thawing simplified 499
peculiarities of oocyte freezing and thawing 501
Oocyte vitrification 187
materials and methods 188
results 189
Open-pulled straw 272
Organ dose 163
Osmotic equilibration 196
Ovarian and testicular tissue 432
Ovarian tissue banking 207
age of the patient 208
difficulties 208
indication 208
protocols 208
risk of ovarian metastasis 208
strategies for preserving female fertility 209
types of cancer 208
Ovarian tissue cryopreservation 232
application 236
autotransblantation 236
in vitro culture 238
xenotransplantation 238
cryopreservation of intact
ovary 239
evaluation of cryopreserved ovarian tissues 236
in vitro culture 236
LM and TEM analyses 236
xenotransplantation 236
future 240
indications 232
autoimmune diseases 233
cancers in adults 233
cancers in children 233
methods 233
influencing factors 236
slow freezing 233
vitrification 234
procedures 233
tissue preparation 527
Ovarian tissue freezing 526
collection of ovarian tissue 527
cooling program 531
cryopreservation of the
whole ovary 526
cryoprotectant preparation 529
equilibration procedure 529
histological analysis 529
indications for ovarian
cryopreservation 527
peculiarities of ovarian tissue
freezing 531
role of vitrification in human
ovarian tissue 526
seeding protocol 530
tissue preparation 527
Ovarian tissue thawing 533
ovarian tissue thawing:
simplified 534
sites of transplant 533
P
Packaging devices 100
capillary-based devices 101
CBS HSV kit 102
classic straws 101
cryotubes/cryovials 100
glass ampules 100
high security straws 101
packaging devices for
vitrification 101
proprietary devices 102
risk analyses 103, 104
controlled rate freezing packaging devices 104
vitrification packaging devices 105
sanitization of packaging devices
contramination of liquid nitrogen 102
controlled rate freezer 102
cross-contamination 102
Packaging systems 121
pH meter 484
Physics of freezing 146
contamination 155
cryopreservation protocols 156
embryos 157
peripheral blood mononuclear cells 156
spermatozoa 157
effects of freezing on cells 148
procedures for ICE nucleation 152
special case of embryos and oocytes in straws 153
reproducibility of protocols 158
supercooling and cell survival 151
thawing and post thaw
handling 156
Polscope technology 242
azimuth 244
birefringence 242
retardance 243
the oosight system 244
Posthumous reproduction 431, 439
cases 442
France: parpalaix versus Cecos, 1984 443
United Kingdom Evans versus the United Kingdom (application no. 6339/05) 443
United Kingdom versus Diane blood (1997) 444
USA Davis versus Davis (1990) 443
USA Hecht versus Superior Court 1993 442
conflicts between the embryo and the mother 436
control over embryos 436
disposition of unused embryos 434
grounds for extended storage 433
Indian scenario 444
infertility treatment act 434
ownership rights for gametes and
embryos 436
posthumous use of gametes and
embryo 434
regulation of ART 438
India 439
international 438
USA 438
the status of the embryo and of
gametes 435
the theory of progressive legal
protection 435
use and control of genetic
material 433
Process mapping 76
Product family overview 106
Progress in cryobiology 252
Properties of nitrogen 3
Protocol for the using HSV kit 114
resources 117
simplified procedure chart 116
thawing 115
Protocols for using HS straws
embryos 108
semen 108
simplified procedure chart 110
embryos 111
sperm 110
Q
Quality assurance 69
Quality control 69, 80
internal quality control and quality assurance 70
instruments and equipment 71
lab conditions and
environment 73
lab personnel 72
maintenance and quality
check 72
media and reagents 70
mouse embryo bioassay 71
sperm survival test 71
technical accuracy and error 73
third party services 71
uncertainty of measurement 74
monitoring protocols
process mapping 76
standard operating
procedures 78
user requirement
specification 77
practical implementation
frequency of assessment 74
planning strategy 74
quarterly-participation of
external quality
assessment 75
total quality management 69
cost control 70
essential principles 70
role of customer expectation 70
Quality cycle 70
Quarantine tank 133
R
Recrystallization 128
Regenerative medicine 391, 400
S
Safety from HIV under
crypreservation 319
materials and methods
experimental protocols 320
supplies and reagents 320
results
control straws 320
PVC straws 320
safety of cryopreservation straws
IR straws 321
PETG straws 321
Sealed opened straws 273
Seeding 126, 127, 156
Semen banking 345
acceptability of frozen donor samples
fresh vs frozen semen 349
cryobiology of sperms 346
selection of donors 347
ethical aspects of therapeutic donor
insemination 350
historical perspective 345
issues in regulation of donor insemination 350
aspects of regulation in donor
insemination 351
commissions, committees 351
current level of regulation 351
laboratory screening 352
quarantine 352
regulatory options for donor
insemination 350
semen samples 352
sperm bank 352
protocols of laboratory
techniques 348
Semen cryopreservation 46, 78
accreditation and ISO standards 86
benchmarking 85
document control 80
inventory and record
maintenance 85
legal issues 86
method 79
principle 78
quality control 79
reagents 79
risk management 83
risk management and laboratory safety 81
organizational issues 82
resource issue leading to high
risk situations 82
risk management issues 83
staffing issues leading to high
risk situations 82
safety of lab personnel 84
specimen 78
tools and solution in cryobiology 83
tools for TQM cryopreservation 80
analysis 81
root cause analysis and
troubleshooting 81
trouble shooting and root cause 81
Semen thawing 473
assessment of post-thaw
fertility 473
semen thawing simplified 474
Shipping 134
Slow freezing 124
Slush nitrogen 9, 142
Solid-surface vitrification 143
Specimen identification 97
auditing 98
inventory systems 98
labeling 97
Sperm bank 352
Sperm counting device 469
Sperm cryobiology 26
Sperm cryopreservation 211
effect of storage temperature 212
specific situations 214
absence of the partner 215
intraoperative sperm harvesting 215
non-obstructive azoospermia 214
obstructive azoospermia 214
patients with cancer 214
Sperm freezing 40
media 469
Spermatogenesis 536
Spindle apparatus 245
ensuring oocyte maturation prior to
freezing 248
gross spindle morphology 246
spindle negatives 245
spindle orientation 247
spindle retardance 246
Standard operating procedures 78
Stem cells 400
sources 401
umbilical cord blood 401
types 400
adult 401
embryonic 400, 401
Steps of cryopreservation 455
cross infection in the semen
banks 470
essentials of freeze thaw cycle 462
cooling and warming rates 463
freezing 463
packaging of semen after addition of cryopreservative 462
plastic straws 462
specimen glycerolization 462
storage 463
events during freezing 457
events during thawing 457
indications of semen cryopreservation
biochemical and physical aspects of sperm cryopreservation 461
common indications for autologous semen banking 461
principles of cryobiology 460
biological behavior of the
phospholipids membrane of
the sperm 460
changes in valume of sperm
when exposed to
cryoprotectants 460
risk of storing biological materials
at low temperatures 458
sperm freezing medium 464
the disposables /equipment
required for cryofreezing are
enumerated as below 467
the techniques for semen
cryofreezing using liquid
nitrogen vapor cooling 464
Storage and use of ovarian and
testicular tissue 432
Storage temperatures 95
Supercooling 151, 156
T
Temperature measurements 92
Testicular tissue cryopreservation 536
ethical issues 539
freezing technique protocol 538
indications
spermatogenesis and stem cells 536
testicular damage postchemo or radiotherapy 536
options 536
principles of testicular preservation
cryoprotectants 537
methods of cryopreservation 538
safety of the cryofreezing
procedures 537
Testicular tissue cryopreservation 536
ethical issues 539
freezing technique protocol 538
indications
spermatogenesis and stem cells 536
testicular damage postchemo or radiotherapy 537
options 536
principles
cryoprotectants 537
methods of cryopreservation 538
safety of the cryofreezing
procedures 537
Testicular/epididymal sperm
freezing 354
cryobiological principles 355
physiology and spermato-genesis 354
sperm freezing techniques 356
sperm retrieval techniques 354
Thawing kit 251
Thawing procedure 258
diluting 259
preparation of media 258
thawing 258
washing 259
Thermal shock 90, 124
Thermodynamics 453
Total quality management 69
Trehalose 39
Tri-laminate zona pellucida 248
U
Umbilical cord blood 417, 427
Undercooling 156
Universal contamination 133
V
Validation studies 106
biocontainment/sanitary
safety 106
Validation studies 112
cooling rate study 112
embryo survival 114
Vitrification 39, 57, 94, 126, 140, 156, 194, 200, 223, 254, 266, 276, 453
alternative to slow freezing 276
aseptic vitrification 279
benefits and disadvantages 224
decreased chilling injury 228
decreased toxicity of
cryoprotectants 224
increased cooling and warming
rates 224
potential danger of disease
transmission 228
carrier system for vitrifying
procedures 201
common carrier systems 272
composition of solutions 58
non-permeating 59
permeating 58
conditions for achieving a vitrified state
first condition 277
second condition 277
factors influencing vitrification 267
buffering solutions 267
cryoprotective agents 267
disaccharides 267
macromolecules 268
future development and safety 61
high concentrations of CPs 278
history and definition 266
importance of cooling rates 201
limitation 274
method and protocol 201
procedure of ultra-rapid
cryopreservation 62
renewed interest 276
re-warming the embryo 278
slow deep-freezing and
vitrification 278
slow freezing versus
vitrification 266
technique of vitrification 268
cryo-solutions 268
embryo/blastocyst
vitrification 269
loading the oocytes in the
cryotip 269
oocyte vitrification 268
thawing 270
materials (expendables) 270
thawing from cryotip 270, 271
warming solutions 270
toxicity of cryoprotectants 60
types of cryoprotectants 201
vitrification of blastocysts 280
vitrification of embryos at D3 280
vitrification of human embryos 279
vitrification of zygotes 279
Vitrification devices 304
material and methods 305
results 306
deep-freezing kinetics 307
effect of the deep-freezing
medium 306
thawing kinetics 308
temperature measurements 305
deep-freezing kinetics 306
effect of the deep-freezing
medium 305
thawing kinetics 306
Vitrification of human embryos 369
Vitrification principles 23
Vitrification procedure 255
equilibration 258
handling tools 257
preparation of media 255
preparation of vitrification
container 255
storage 258
vitrification 258
Vitrification-kit 251
W
Warming/thawing 128
Water molecule 11
application in cryobiology 21
crystal structures of ice 13
hexagonal 14
tetrahedral 13
crystalline phases of ice 14
formation 12
H2O molecule in liquid water 16
H-bonds in liquid water 16
molecule in ice 13
properties 13
properties of liquid water 16
latent heat 19
physical 17
×
Chapter Notes

Save Clear


1FROZEN LIFE2
3FROZEN LIFE
A Manual of Cryobiology for Assisted Reproduction and Stem Cells
Editor Pankaj Talwar Associate Professor Department of Obstetrics and Gynecology Assisted Reproductive Technologies Specialist Trained in Human Embryonic Stem Cell Biology Army Hospital (Research and Referral) Delhi Cantt 110 010, India
4
Published by
Jitendar P Vij
Jaypee Brothers Medical Publishers (P) Ltd
Corporate Office
4838/24 Ansari Road, Daryaganj, New Delhi 110 002, India, Phone: +91-11-43574357, Fax: +91-11-43574314
Registered Office
B-3 EMCA House, 23/23B Ansari Road, Daryaganj, New Delhi 110 002, India
Phones: +91-11-23272143, +91-11-23272703, +91-11-23282021,
+91-11-23245672, Rel: +91-11-32558559 Fax: +91-11-23276490, +91-11-23245683
Branches
North America Office
1745, Pheasant Run Drive, Maryland Heights (Missouri), MO 63043, USA, Ph: 001-636-6279734
Central America Office
Jaypee-Highlights Medical Publishers Inc., City of Knowledge, Bld. 237, Clayton,
Panama City, Panama, Ph: 507-317-0160
Frozen Life
© 2009, Jaypee Brothers Medical Publishers
All rights reserved. No part of this publication should be reproduced, stored in a retrieval system, or transmitted in any form or by any means: electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the editor and the publisher.
First Edition: 2009
9788184486810
Typeset at JPBMP typesetting unit
Printed at Ajanta Press
5Dedicated to
My father Late MP Talwar and my mother Madhu Talwar without their blessings such monumental work would not have been possible.
To
My wife Dr Neetu Talwar and sons Pratik and Arjun for giving unfailing support and encouragement during compilation of the book. It was their constant motivation which made me move ahead in trying times.
To
All my unknown patients who visited me for treatment with utmost faith and gave me opportunity to learn from their illness6
7Contributors
12
13Foreword
It is indeed a great pleasure to write a foreword for this book on cryobiology. Human reproductive cryobiology is rapidly developing subspecialty of Assisted Reproductive Technologies.
Since the first report of human pregnancy and the first live birth following cryopreservation two decades ago, the techniques has developed as a reliable science and is carried out regularly at all ART laboratories.
Combined with tailored patient preparation protocols, thawed embryo transfer cycles have become relatively successful. The transfer of cryopreserved embryos represents around 20% of all transfers worldwide.
Recently, the focus is shifting to the vitrification technique which involves ultrarapid cooling of the reproductive cells resulting in glass formation. A lot of researches are going on in development of ideal oocyte cryofreezing protocols. If perfected such technique would be an important technology to help unmarried women who are suffering from malignancy and require treatment.
The book has covered these topics at length and would be ideal compendium for any ART manual.
I am happy that Lt Col Pankaj Talwar has taken this initiative to edit the textbook of Frozen Life. The book has been written in a concise and systemic manner and readers can easily assimilate the subject and practise it.
Punita Arora pvsm, sm, vsm
Consultant, Obstetrics and Gynecology (Armed Forces)
Ex-Director General Medical Services and Ex-Commandant and Director
Armed Forces Medical College
Pune 4014
15Foreword
I am happy to write a foreword to this textbook of cryobiology by my student Lt Col Pankaj Talwar. This is probably the 1st book ever published dealing entirely with the principles of cryobiology and its application in Assisted Reproductive Technologies.
The main aim of cryopreservation for human oocytes and embryos is to reduce damage caused by intracellular/extracellular ice crystal formation. To achieve this, it is important to consider the behavior of intracellular water at subzero temperatures. Two basic techniques have been developed for reproductive cells cryopreservation: controlled slow-rate freezing protocols and rapid freezing protocols such as vitrification.
These techniques may be applied to cryofreezing of oocytes, spermatozoa, embryos, genital tissue, umbilical cord blood cells and embryonic stem cells. All these techniques and protocols are thoroughly covered in this book. The protocols have been well elucidated and deliberated.
The editor has covered the process of cryobiology step by step at length in the last section of the book. This will reduce the learning curve of the embryologists and clinicians. These protocols encompass the techniques being followed at Army Hospital (Research and Referral), New Delhi, India.
I congratulate the editor for bringing so many researchers, clinicians and reproductive biologists under the one roof and compiling this textbook. I am sure this book will help all the medical personnels working in the field of Assisted Reproductive Technologies and related specialties to understand the topic better and put it to practical use.
Professor and Head
Department of Obstetrics and Gynecology
Director, Assisted Reproductive
Technologies Centre, Army Hospital
(Research and Referral)
Delhi Cannt 110 010
India16
17Preface
Cryobiology is a rather interesting but complex subspecialty of Assisted Reproductive Technologies. This branch has been always considered as an appendage of ART and clinicians have been somewhat sceptical about accepting the concept of creating icecream babies. The protocols are easy but the physiology and thermodynamics of the procedure are not clear to majority of us. This scares us from accepting this technology.
I am endeavoring to simplify the gray areas of cryobiology with lucid articles and videos pertaining to all aspects of reproductive medicine and stem cell biology.
As the embryo culture conditions are improving, more number of embryos are available to the embryologist for cryofreezing. With advent of single embryo transfer, the Assisted Reproductive Technology centres have been encouraged to adopt cryobiology techniques in their day-to-day practice.
The advent of oocyte freezing for fertility preservation along with the advances in the fields of ovarian tissue and testicular tissue banking has generated enormous interest in this field.
Till date there has been a scare in minds of reproductive biologists who prefer fresh embryo transfer in the IVF cycles. Prior experiences with poor cryosurvival of the gametes and embryos and compromised implantation rates were not encouraging.
Better protocols and development of less toxic cryoprotectant solutions have led to resurgence of the subspecialty which had taken backseat due to poor outcomes in past few years.
Cryobiology has played pivotal role in cryopreservation of hematopoietic stem cells and embryonic stem cells. Reproductive biologists are slowly shifting the focus of research toward vitrification which is cheaper, quicker and easier technology.
The aim of the book has been to bring together researchers, clinicians and embryologists on one platform to share their views on various aspects of cryobiology.
A wealth of information pertaining to basics of cryobiology, thermodynamics of cryofreezing, cryoprotecants along with newer protocols for vitrification of embryos, oocytes, etc. have been thoroughly covered. We have also covered at length umbilical cord blood banking and cryobiology of embryonic stem cells.
Efforts have been made to present to the readers the protocols using lots of pictures so that they can closely associate with the techniques and easily assimilate them.
The subject is vast and has been covered in 53 Chapters and Appendices. Some amount of repetition is unavoidable while compiling such monumental work.
Please feel free to communicate with me if you need any clarifications or have any suggestions.
Pankaj Talwar18
19Acknowledgements
I would like to thank all my teachers, colleagues for their constant motivation and encouragement.
My sincerest thanks to Col RK Sharma and Lt Gen (Mrs) Punita Arora who constantly held my hand in this humble spiritual journey and gave me strength to move on in the face of all adversities.
I am grateful to Lt Gen Y Singh PVSM, VSM, PHS, DGAFMS and Sr Col Comdt, Lt Gen NK Parmar AVSM, VrC, VSM, PHS, DGMS Army and Col Comdt, Lt Gen OP Mathew AVSM, SM, Col Comdt and Commandant Army Hospital (Ramp;R), for permitting me to take on this monumental work. Without their constant support the book would not have been in our hands today.
I am also grateful to Prof Itskovitz-Eldorj or Amit M and team from technion, Israel, for teaching me the intracacies of embryonic stem cell biology.
I would like to thank my colleagues Col BS Duggal and Col BK Goyal for the guidance and constant motivation.
The work would not have been possible without unflinching support of Shri Jitendar P Vij (Chairman and Managing Director), Mr Tarun Duneja (Director-Publishing), Mr Subroto Adhikari, Mr KK Raman, Mr DC Gupta, Mr Manoj Pahuja, Mrs Y Kapoor and Mrs Niti of M/s Jaypee Brothers Medical Publishers (P) Ltd., New Delhi.
I thank Trivector Scientific Pvt. Ltd., Dr H Ingolf Nilesen and Dr Lars Johanson, consultant embryologists (MediCult, Denmark) for their suggestions and constant support in compilation of the manuscript.
I would like to thank IMV India Ltd., Alex Deroubaix of Air Liquid, Alain Ehrsam, Philippe Clairaz, Ms Beatrice Ledos and Ms Melanie lelogof CBS France, Intermedic and Cook International (Australia), FertiPro (Belgium), vitrolife for support and permission to utilize the videos on vitrification for educational purposes.
I am thankful to Dr Sarabpreet Singh, Mr Parikshit Singh and Mr GS Pillai who spent late nights with me while compiling pictures for of this book.
I humbly acknowledge that this dream would not have been possible until all the contributors rendering me their unflinching support and motivation. The journey from inception of the book to its publication was rough but I was always supported by my friends and my patients.
20Disclaimer
This manual presents an understanding and an outlook of the current scientific advances in the field of reproductive and stem cell cryobiology. This is an emerging field and is changing every day. The information presented in this book may not be considered as exclusive methodology of cryobiology techniques; rather, it is intended to be a basic guideline for the clinicians and embryologists.