Surrogacy

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Embryology 101

• Dr. Samit Sekhar

• Executive Director and Embryologist

• Kiran Infertility Centre Pvt. Ltd

• Basics of embryo development in an IVF laboratory.

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Embryology in Greek means "the unborn, embryo)
is the science of the development of an embryo
from the fertilization of the ovum to the fetus
stage.
In a routine ART (Assisted reproductive
Technology) setting we create embryos by fusing
sperm from the male partner with the egg of the
female partner.
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Treatment Option 1
In vitro fertilisation (IVF) commonly known as
test tube baby as we all know is one of the most
significant advances in the field of modern
medicine. The first IVF baby Louis Brown, born in
England in 1978 was the result of more than a
decade of research by Dr. Patrick Steptoe and
Robert Edwards. They essentially changed the
rules for how people can come into the world.
Conception was now possible outside the body in
a petri dish. The technique has resulted in the
births of millions of babies worldwide, many in
multiple births.
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• Initially ivf was developed for women with tubal
  disease but now it is the treatment of choice for
  several other causes of infertility,since its
  introduction all the steps of ivf have been
  improved upon which has resulted in continuously
  rising success rates.
• IVF has also provided a platform for the
  development of other treatments including egg
  donation, Gestational surrogacy and
  preimplantation diagnosis

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Steps of IVF

• 1. Ovulation Induction is as per the protocols
  based on several factors of the female partner
  and might be a Down regulated or Antagonist
  cycles.
• 2. Oocyte retrieval is performed under vaginal
  ultrasound guidance. After the vaginal ultrasound
  is placed in the vagina and the ovarian follicles
  are located a needle is directd through the back
  wall of the vagina and directed into the ovarian
  follicles.
• The fluid is aspirated and then examined by the
  embryologist to identify the microscopic egg -
  oocyte surrounded by granulosa cells (cumulus).
  During normal fertilisation the acrosome of the
  sperm releases enzymes which disperse the cumulus
  cells thereby allowing the sperm to penetrate and
  fertilise the oocyte.
• Once the eggs are retrieved they are placed in
  culture media with added nutrients and then
  placed in the incubator. The procedure is
  performed under a general Anesthesia and
  generally takes less than 10 to 15 minutes to
  complete. There are minimal complications(lt1)

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Steps of IVF

• 3. Oocyte Insemination After the male partner
  produces the sperm sample by masturbation, the
  sperm concentration and motility are assessed. If
  found adequate the sperm preparation is done to
  collect the most motile sperm. A total of nearly
  50,000 motile sperm is placed around the eggs in
  a culture dish which is then placed in the
  incubator
• Denudation is the process by which following
  oocyte insemination the surrounding cumulus is
  removed to check for signs of fertilisation using
  denuding pipettes. After a while the nuclei unite
  and the embryo will start to divide
• 4. Embryo Culture The embryos are cultures in a
  petri dish inside specially designed incubators
  using nutritive media.

Oocyte Insemination
A fertilised Egg
Petri Dish
Incubators
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• 5. Embryo Transfer usually performed 72 hours
  after the egg retrieval, generally 2 to 4 embryos
  are transferred into the uterine cavity under
  abdominal ultrasound guidance.

ET Catheter
The recommended number of embryos to transfer is
determined by the womens age, cause of
infertility, previous pregnancy history and other
factors including the acceptance of a twin
pregnancy. During transfer a full bladder
creates an opportunity so that uterus can be
clearly visualised and an echogenic catheter is
easily seen as it is passed through the cervical
canal and into the uterine cavity. The catheter
is placed about 2cms away from the top of the
cavity where the embryos are released.
Ultra Sound ET
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ICSI - Intra Cytoplasmic Sperm Injection

• ICSI - Intra cytoplasmic sperm injection is used
  in cases of male factor infertility or in cases
  where standard ivf results in lt40 fertilised
  embryos from previous ivf cycles or there is no
  fertilisation at all in a previous ivf cycle.
• ICSI involves the injection of a single sperm
  directly into the oocyte.
• Fertilisation rates using icsi are between 70 to
  80.
• Males with severe oligospermia(lt5 million/cc)
  should have a chromosomal karyotype performed
  since they are at a greater risk of chromosomal
  abnormality.

ICSI
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Related procedure to IVF

• 1. FET (frozen embryo transfer) - extra embryos
  can be cryopreserved by standard freezing or by
  vitrification and can be transferred in the next
  cycle after spontaneous ovulation or creation of
  an artificial endometrium with estrogen and
  progesterone.
• The success rates using frozen embryos is
  slightly lower than fresh embryos. The main
  advantage of freezing embryos is that ovulation
  induction need not be given to the donor again.
• Studies have shown that there are no increased
  risk of congenital anomalies in infants born
  following the use of frozen / thawed embryos.
• 2. Epididymal Sperm Aspiration
• In some cases of Azoospermia, sperm is produced
  but are not present in the ejaculate as a result
  of Obstruction because of infection, congenital
  conditions or a previous sterilisation.
• In these cases aspiration of epididymal sperm or
  testicular sperm by a specialist is carried out
  and the resultant sperm is used for ICSI(image)
• The sperm aspiration maybe carried out on the day
  of oocyte recovery or prior to ivf cycle and the
  samples maybe frozen.

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Related procedure to IVF

• 3. Assisted Hatching- is a procedure in which the
  zona pellucida, the outer membrane surrounding
  the embryo is thinned by the addition of a dilute
  acidic solution and mechanically broken down
  based on the theory that implantation failure
  maybe the reslt of failure of the embryo to hatch
  out from within the zona pellucida,commonlyused
  for women over 35 years of age or after several
  unsuccessful transfers. Hatching may be done
  using chemicals or by laser.
• 4. Blastocyst Culture- The blastocyst stage of
  embryonic development occurs just prior to
  implantation. The blastocyst is an embryo made up
  of 80 to 100 cells and reaches this stage 5 to 6
  days after egg retrieval. Usually after day 3 the
  embryos need to be transferred into a special
  Blastocyst medium for them to grow to Blastocyst
  stage.generally 40 to 50 of embryos develop
  till Blastocyst stage, the Advantages of
  Blastocyst Allows for selection of best embryos.
  Reduces the chances of twins or triplets.
   Disadvantage - Monozygotic twinning(3)

Blastocyst Embryo
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Related procedure to IVF

• 5. PIGD has turned out to be a big boon for
  couples who were at a risk of having a child born
  with a genetic or a chromosomal disorder,the only
  options earlier were chorionic villous sampling
  or amniocentesis. These are invasive and not the
  first choice anymore.
• In PIGD a single blastomere is removed from the
  embryo and tested prior to transfer. It was
  carried out first in 1990 for a couple who were
  at risk of having a child born with cystic
  fibrosis.
• Commonly trisomy 13/18 and 21 and common genetic
  disorders are tested however with the
  availability of more and more genetic probes the
  increase in demand will only grow further.
• 6. Oocyte freezing is another emerging
  technology.the oocyte is more sensitive to the
  cryopreservation process than the embryo because
  of the high water content in the egg which
  predisposes it to ice crystal damage
• Indications-women undergoing cancer treatment to
  preserve their fertility,for younger women who
  are career minded and want to preserve their
  fertility for future.
• Couples undergoing ivf also sometimes prefer to
  freeze eggs instead of embryos because once their
  family is complete is would be emotionally easier
  to discard frozen eggs instead of embryos.

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Oocyte scoring and embryo grading

• Oocyte scoring
• The factors that are included in the evaluation
  of oocyte quality were
• oocyte cytoplasmic dysmorphisms, extracytoplasmic
  dysmorphisms
• and the oocytecoronacumulus complex.
• Assessing fertilization and zygotes (Day 1)
• Assessment of fertilization is straightforward,
  as a fertilized
• oocyte should have two pronuclei and two polar
  bodies.
• Cleavage-stage embryo scoring (Day 2 and 3)
• A. Top quality
• B. Good quality (not for elective single embryo
  transfer)
• C. Impaired embryo quality
• D. Do not recommend to transfer

Fertilized Oocyte
Oocyte Scoring
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• Embryo quality evaluation is of prime importance
  in order to sustain a successful IVF / Surrogacy
  program.
• Standard assessment by an embryologist relies on
• Cell numbers
• Fragmentation
• Blastomere size
• Cytoplasmic anomalies
• Compaction
• Cleavage stage embryos range from the 2-cell
  stage on day 1 to the compacted morula composed
  of 816 cells day 3 and 4. The number of
  blastomeres on day 2 or 3 is used as the primary
  criteria for embryo assessment.
• Good quality embryos (A grade) must exhibit
  appropriate division and synchrony of division.
  In normal embryos, cell division occurs every
  1820 hours. Embryos dividing either too slow or
  too fast may have metabolic and/or chromosomal
  defects. It is also pertinent to note that the
  environment in the specific laboratory, such as
  culture media and temperature,PH etc influences
  the quality and grading of embryos.

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Stages of Embryo Development
2 Cells
5 Cells
4 Cells
3 Cells
6 Cells
8 Cells
7 Cells
9 Cells
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• Each blastomere should have a single nucleus.
  Multinucleation has been described to be
  associated with genetic disorders of the embryo
  and has been associated with an increased
  abortion rate Multinucleation can be evaluated on
  Days 1, 2 and 3 of development.
• Grade-A embryo characteristics blastomeres of
  equal size, little or no fragmentation, and a
  zona pellucida that is not extremely thick or
  dark in appearance.
• Grade-B embryo characteristics blastomeres of
  equal size, minor cyotplasmic fragmentation
  covering lt 10 of the embryo surface.
• Grade-C embryo characteristics blastomeres of
  distinctly unequal size and moderate to
  significant cytoplasmic fragmentation covering
  gt10 of the embryo surface.

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• A clear homogeneous cytoplasm is a sign of
  normal cleavage stage embryo. The presence of
  multiple vacuoles is a sign of a poor quality
  embryos
• Compaction- After the embryo reaches the 8-cell
  stage, the blastomeres begin to show an increase
  in cell to cell adherence and this is known as
  compaction and the process continues until the
  boundaries between the cells are barely
  detectable . If some of the blastomeres are
  excluded from this compaction process, the embryo
  may have a reduced potential for becoming a
  normal blastocyst

Compaction
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• Blastocyst transfer
• Day 5 or day 6 embryos are called blastocyst
  stage embryos. The blastocyst stage is the stage
  of development that an embryo must reach before
  it can hatch and implant in the uterus
• Blastocyst embryos consists of a blastocoel which
  causes the embryo to start to expand and increase
  in overall size. Blastocyst embryos have two
  distinct parts, the inner cell mass (these cells
  develop and become the fetus), and the
  trophectoderm cells (these cells will develop
  into the placenta).  
• The blastocyst is given a grade based upon the
  three main components of the blastocyst embryo.

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• Expansion Grade
• Early Blastocyst the blastocoel filling more
  than ½ the volume of the embryo, but not
  expansion in overall size compared to earlier
  stages.
• Blastocyst the blastocoel filling more than ½
  the volume of the embryo, with slight expansion
  in overall size and notable thinning of the zona.
• Full Blastocyst a blastocoel filling more than
  50 of the embryo volume and overall size fully
  enlarged with a very thin zona.
• Hatching Blastocyst The trophectoderm has
  started to herniate through the zona.
• Fully Hatched Blastocyst Free blastocyst fully
  removed from the zona.

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• Inner Cell Mass
• A     Tightly packed compacted cells.
• B     Large, loose cells
• C     No ICM distinguishable
• D     Cells of the inner cell mass appear
  degenerative
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• Trophectoderm Grade
• A     Many healthy cells forming a cohesive
  epithelium
• B     Few but healthy cells, large in size
• C     Poor, unevenly distributed cells. Many
  appear as few cells squeezed to the side
• D     Cells of the trophectoderm appear
  degenerative embryo surface.

Hatching Blastocyst
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• Embryo freezing
• Step 1. consent
• Step 2.  Screening for both people who provided
  the eggs and sperm used to create the embryos for
  infectious diseases such as HIV, Hepatitis B C.
• Step 3. During treatment, the unused embryos are
  frozen and then stored in tanks of liquid
  nitrogen. A liquid called a cryoprotectant is
  added to protect the embryos during freezing.
• In the standard freezing method, embryos are
  slowly frozen down to -196 degrees celcius.
• vitrification. is a fast freeze process the
  embryo undergoes instantaneous glass-like
  solidification without the damaging formation of
  ice crystals (which can occur with the standard
  method of freeze)

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How long can the embryos be stored?

• The normal maximum period that frozen embryos can
  be stored is ten years. This may be extended
  depending on the medical circumstances of the
  woman undergoing treatment, her partner and/or a
  donor.
•  

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