Chronology of IVF-ICSI Research in INDIA

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In-Vitro Cell Technology in India Origin,
Evolution and New frontiers
Dr. Sudarsan Ghosh Dastidar, MD GD Institute for
Fertility Research Kolkata, India
HEAL Conference, India International Centre , New
Delhi, 23rd September 2009
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Assisted Reproductive Technology (ART) - till now

• Manipulation of human cells (egg and sperm) out
  side the body, i.e., In-Vitro, started with the
  breakthrough of test tube baby technology in
  1978.
• Two main modalities
• IVF
• ICSI
• The advanced laboratory set up and technology
  required for this may be the basis for embryonic
  stem cell culture and future cell based therapy.

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Chronology of IVF-ICSI Research in INDIA

• 1978 Dr. Subhas Mukherjee his team.
  Birth of Durga.
• 1982-1986 Kolkata-Dr. B. N. Chakravorty
• Dr. S. Ghosh Dastidar
• Birth of Imran.
• 1983-1986 Bombay- Dr. I. Hinduja
• Dr. Anandkumar
• Birth of Harsha ( July 1986 )
• 1992 Kolkata- 1st GIFT Baby
• Dr. S. Ghosh Dastidar Dr. K
  Ghosh Dastidar
• 1995 Kolkata- 1st ICSI ZIFT Baby
• Dr. S. Ghosh Dastidar Dr. K
  Ghosh Dastidar
• 1995 Mumbai- ICSI Baby
• Dr. Firuza Parikh

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Dr. Subhas Mukherjee The pioneer of IVF Research
in India.Responsible for inducting me into the
emerging field of In-Vitro Fertilization and
related science.
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World Congress on IVF, Helsinki, Finland, 1984
World Congress on IVF, Istanbul, 2005
Cornell Medical Center, USA Dr. Bedford and
myself, 1987
Prof. Struart Campell, myself and Dr. Kakali
Ghosh Dastidar
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Our Initial Struggle (1982)

• The story of how we developed IVF in India
  (Kolkata) is one of Herculean struggle. It is
  necessary to narrate many personal details which
  are vital to illustrate how breakthroughs in
  creative science really happen, specially in a
  country like ours.
• Our challenges
• Non existent infrastructure for IVF related cell
  culture in India. Hence I had to prepare
  tissue culture media from scratch, using the bare
  minimum resources available in Kolkata
• Frequent power cuts in 1981-82 standby generator
  was must
• There was no computer and obviously no internet
  thus there was almost no literature/publication/jo
  urnals available in Kolkata
• The most challenging job was to develop a tissue
  culture system which would produce 5 Co2 in air
  and 95 relative humidity atmosphere since we did
  not have the modern computerized Co2 incubator.
• Though it may sound extraordinary to researchers
  today, one basic challenge was to first identify
  how the human egg looked under the microscope,
  BECAUSE THERE WAS NO PHOTOGRAPH/PICTURE available
  in any book/journal/internet

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Learning by Trial Error

• During 81-82 we had no knowledge even about
  identification of human egg or the associated
  laboratory procedures for IVF. Thus we had to
  start from scratch.
• I was entrusted with the responsibility to
  develop basic IVF laboratory aspects like
  Preparation of tissue culture media, Oocyte
  identification sperm preparation
  fertilization embryo-culture.
• The only basic knowledge I possessed was of a
  theoretical nature, and came from my discussions
  with Late Dr. Subhas Mukherjee.

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MAJOR CHALLENGES IN THE EARLY PART OF OUR
RESEARCH

• Prof. B. N. Chakraborty was involved in
  developing the clinical aspects like patient
  selection, ovarian stimulation and most
  importantly, oocyte retrieval and finally embryo
  transfer (ET).
• I was entrusted to develop the embryo culture
  laboratory and then the laboratory methods. My
  major challenges were
• To identify human oocyte from follicular fluid
  aspirate.
• To device some system to produce 5 CO2 in air
  atmosphere for IVF and embryo culture since we
  did not have modern CO2 incubator.
• To capacitate spermatozoa an absolute necessary
  step to achieve successful fertilization of
  oocyte.
• To prepare tissue culture media preparation PH
  and osmolarity control of media. Prof. Subir
  Dutta, an eminent pathologist of Kolkata extended
  logistic and technical support.
• To learn manipulation of human oocyte under
  stereo microscope.
• Thus, I was a self trained IVF laboratory expert!!

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1982-83
A typical day

• Morning 5 6 a.m. cleaning of laboratory,
  final preparation of media and other lab
  protocols
• 7-9 a.m. basic quality control, preparation for
  egg recovery.
• 1030 a.m. to late evening - I used to carry the
  test tube containing follicular fluid in a
  thermos flask to a laboratory in far away
  Jadavpur University and later, to Indian
  Statistical Institute, where I was desperately
  trying to identify the oocytes under stereo
  microscope (no one in Calcutta then even knew
  their appearance !)
• However, soon we realized that in order to
  achieve pregnancy by IVF we needed a laboratory
  close to the clinic where oocyte retrieval was
  being done. Otherwise the temperature and pH of
  the egg-containing follicular fluid was being
  altered in this long transit.
• Thus, I set up Calcuttas first IVF laboratory
  (very primitive though!) in my study room at
  79/28, A.J.C. Bose Road, Kolkata - 14.
• Some days I used to finish work at 12 night or
  even later.

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Humble Beginnings
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and, the modern IVF lab !
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The improvised IVF embryo culture system devised
by me in 1983

• A pre-requisite for IVF is perfectly controlled
  environment for cell culture, currently provided
  by state-of-art incubators as shown in the
  previous slide.
• During 1982-86 we did not have modern incubator -
  an absolutely essential equipment for human IVF.
• Thus I tried to improvise several alternative
  simple system with locally available materials.
• Finally I was able to develop a system to
  produce 5 CO2 in air atmosphere and
  approximately 85 relative humidity.

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Improvised embryo culture system developed by me
using 5 CO2 in air environment (1982)
Modern CO2 Incubators used in our center
currently
5 CO2 in air
Test tube containing egg in culture media
Water in petri dish
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Initial breakthrough in IVF (1982-1984)

• After many failures, I achieved fertilization of
  oocyte and its subsequent cleavage to 6-8 cell
  stage in June/July 83.
• Following embryo transfer, a pregnancy occurred.
  (September 83). Unfortunately, this pregnancy
  ended in abortion at around 10 weeks.
• It was a major break through indicating the
  feasibility of IVF as a clinical method to treat
  tubal factor infertility.
• Dr. B. N. Chakraborty and myself jointly
  presented this data in 3rd World Congress on
  IVF, Helsinki, Finland, May 1984.
• This was the 1st report of successful IVF and
  embryo transfer resulting in pregnancy in human
  from India in any World Congress. (May84)
• Ref. Our Experience of IVF in India Chakraborty
  B.N. and Ghosh Dastidar S. Congress book 3rd
  World Congress on IVF, Helsinki, Finland, May 1984

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Microphotographic Documents of oocytes taken by
me during 1982-1983
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Offshoot of IVF Technology novel sperm
preparation and IUI in human infertility

• The technology of sperm preparation in IVF,
  prompted me to venture introducing 100 motile
  sperm in uterine cavity in patients with
  unexplained infertility.
• I was surprised to achieve series of pregnancies
  following such method during 1982-84.
• These were the very first few IUI
  pregnancies in the world.

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Abstract of our paper presented at 5th World
Congress on Human Reproduction Athens, Greece,
1985
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ICSI the major breakthrough in Assisted
Reproductive Technology after IVF

• Till 1993 there was no treatment for severe male
  factor infertility with very low sperm
  count/motility, not even by IVF
• This changed with the advent of Intra Cytoplasmic
  Sperm Injection (ICSI) which means fertilizing an
  egg by micro injection with a single viable
  spermatozoa in the laboratory, first reported in
  1992-93 from Brussels, Belgium.
• I initiated one of Indias first two ICSI
  programs in Kolkata in January 1994, which was
  responsible for the birth of a ICSI-ZIFT baby in
  March 1995.

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Intra Cytoplasmic Sperm Injection
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Establishment of Successful ICSI in Our Center,
1994-1995
Our first ICSI baby in 1995
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Different stages of fertilized egg and embryo
following IVF/ICSI documented in my Lab
4 cell stage
PN Stage
8 cell stage
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What next ?
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Blastocyst embryo
Morula stage
Blastocyst stage
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Human Embryonic Stem Cell (hESC)

• Stem cells are unspecialized. One of the
  fundamental properties of a stem cell is that it
  does not have any tissue-specific structures that
  allow it to perform specialized functions. For
  example, a stem cell cannot carry oxygen
  molecules through the bloodstream (like a red
  blood cell).
• Stem cells can give rise to specialized cells.
  When unspecialized stem cells give rise to
  specialized cells, the process is
  called differentiation.
• Scientists are just beginning to understand the
  signals inside and outside cells that trigger
  each step of the differentiation process.
• The internal signals are controlled by a
  cell's genes, which are interspersed across long
  strands of DNA, and carry coded instructions for
  all cellular structures and functions.

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Historical Landmarks
Scientists discovered ways to derive embryonic
stem cells from early mouse embryos
1981
The detailed study of the biology of mouse stem
cells led to the discovery of a method to derive
stem cells from human embryos and grow the cells
in the laboratory. These cells are called human
embryonic stem cells.
1998
Major breakthrough - identification of
conditions that allow some specialized adult
cells to be "reprogrammed" genetically to assume
a stem cell-like state. This new type of stem
cell, called induced pluripotent stem cells
(iPSCs),
2006
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How are embryonic stem cells grown in the
laboratory
Growing cells in the laboratory is known as cell
culture. Human embryonic stem cells are isolated
by transferring the inner cell mass from the
blastocyst into a plastic laboratory culture dish
that contains a nutrient broth known as culture
medium.
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Pluripotency the God of small cells

• Human Embryonic stem cells, as their name
  suggests, are derived from 4-5 days old surplus
  human embryos produced by IVF centers.
• They self-renew indefinitely in the
  undifferentiated state producing millions of
  cells.
• They are pluripotent, which means they can
  develop into all cells and tissues in the body.
  Have unique regenerative abilities
• The most important potential application Cell
  Based Therapies in
• 1) Diabetes
• 2) Alzheimers disease
• 3) Spinal cord injury
• 4) Stroke
• 5) Heart disease
• 6) Osteoarthritis
• With further research it may be possible to
  understand how cell proliferation is regulated
  during normal embryonic development or during
  abnormal cell division and differentiation that
  leads to cancer.

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The invisible Hands of God the miracle solution
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How are embryonic stem cells stimulated to
differentiate?
Directed differentiation of mouse embryonic stem
cells.
Diseases that might be treated by transplanting
cells generated from human embryonic stem cells
include Parkinson's disease, diabetes, traumatic
spinal cord injury,Duchenne's muscular dystrophy,
heart disease, and vision and hearing loss.
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What are the potential uses of human embryonic
stem cells and the obstacles that must be
overcome before these potential uses will be
realized?

• Studies of human embryonic stem cells will yield
  information about the complex events that occur
  during human development.
• Primary goal - to identify how undifferentiated st
  em cells become the differentiated cells that
  form the tissues and organs. Scientists know
  that turning genes on and off is central to this
  process.
• Serious medical conditions, such as cancer and
  birth defects, are due to abnormal cell
  division and differentiation.
• A more complete understanding of the genetic and
  molecular controls of these processes may yield
  information about how such diseases arise and
  suggest new strategies for therapy.
• Testing of drugs - new medications could be
  tested for safety on differentiated cells
  generated from human pluripotent cell lines.

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Have human embryonic stem cells successfully
treated any human diseases?

• January 23, 2009 - FDA granted clearance for
  Gerons (Califormia based pharma company)
  clinical trial of GRNOPC1 in patients with acute
  Spinal Cord injury the Worlds 1st Human
  clinical trial of Embryonic Stem Cell based
  therapy
• Gerons 2nd HESC product GRNCM1 involves pre
  clinical trial of cardiomyocytes for treating
  myocardial disease.

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In-Vitro Cell Technology in India Origin,
Evolution and New frontiers
IVF
ICSI
Embryonic Stem Cell from Blastocyst
Cell based therapies
Future regenerative medicine

• Our human body as the supreme model of omni
  potent mechanism.
• It appears that in near future practice of
  medicine will be rewritten with inclusion of
  regenerative or restorative medicine.
• The primitive immortal cells that is embryonic
  stem cells appears to have the potential to
  restore the body mechanism from disease process
  by an unique regenerative approach hitherto
  unknown to mankind.

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E  mc2
Electricity
enormous energy
Single atom-------------
?Atom Bomb
Regenerative medicine
Millions or trillions of hESC
Differentiation
Embryonic Stem Cell
-----------
? Ethical issues
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Research Ethics and Stem Cells

• The ethical question which arises is to what
  extent should researchers manipulate the most
  basic processes of human life.
• However, we need to ask ourselves whether the
  potential benefits for treatment of disease and
  improving quality of life outweigh such ethical
  constraints.
• Thus, it would seem that research in embryonic
  stem cell promises much, and also needs
  guidelines.
• Here we should keep in mind that during early
  days of evolution of IVF technology in India, it
  was precisely the lack of constraining regulation
  which permitted such exponential growth.

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Thank you