Procedures for embryo culture.

1. EMBRYO CULTURE AND
MICROMANIPULATION
SUBMITTED TO : SUBMITTED BY :
Dr. Kanchan Sapra Priyanka
Physiologist L-2020-BT-09-M
Dept. Of Vet. Physiology And Biochemistry M.Sc. Biotechnology

2. CONTENT
EMBRYO CULTURE
• What Is An Embryo Culture
• How It Is Performed
• Techniques In Embryo Culture
MICROMANIPULATION
• Introduction
• Widely Used Techniques For Micromanipulation
• Conclusion

3. What Is An Embryo Culture
• Embryo culture is a component of in vitro fertilization where in
resultant embryos are allowed to grow for sometime in an artificial
medium before being inserted into the uterus.

4. HOW IS IT PERFORMED : by 2-ways :-
Artificial culture
Single
culture
Sequential
culture
Autologous
endometrial
coculture

5. ARTIFICIAL EMBRYO CULTURE MEDIA
• It contains glucose, pyruvate and energy providing components.
• Amino-acids, nucleotides, vitamins and cholesterol improve the
performance of embryonic growth and development.
• SINGLE CULTURE : the same culture medium throughout the medium.
• SEQUENTIAL CULTURE : the embryo is sequentially placed in different
medium.

6. AUTOLOGOUS ENDOMETRIAL COCULTURE
• Autologous endometrial coculture is a technique of assisted
reproductive technology . It involves placing a patient’s fertilized eggs
on top of a layer of cells from her own uterine lining, creating a more
natural environment for embryo development and maximizing the
chance for an in vitro fertilization (IVF) pregnancy.

7. HOW COCULTURE IS PERFORMED
• Patient undergoes an endometrial biopsy during which a small piece
of her uterine lining is removed.
• The uterine lining sample is sent to a research lab, where it is treated,
purified and frozen.
• The patient then undergoes a typical IVF cycle and is given
medication to stimulate egg growth in her ovaries.
• The patient’s eggs are retrieved and mixed with the sperm. At this
time, the lab begins thawing and growing her endometrial cells.

8. • Once fertilization is confirmed, the patient’s embryos are placed on
top of her own (and now thawed) endometrial cells.
Over the next two days, the embryos are closely monitored for
growth and development.
The patient’s embryos are transferred into her uterus for
implantation and pregnancy
Coculture can be an effective treatment for patients who have failed
previous IVF cycles or who have poor embryo quality.

9. TECHNIQUES IN EMBRYO
CULTURE

10. • OOCYTE WASH BUFFER
• FERTILIZATION MEDIUM
• CLEAVAGE MEDIUM BLASOCYST MEDIUM
• SPERM BUFFER
• SPERM MEDIUM
• BLASTOCYST CULTURE

11. 1. OOCYTE WASH BUFFER
• On the day of egg retrieval (Day 0), this buffer is used for the retrieval
of the eggs from the ovary. Oocyte wash buffer has an ingredient,
which prevents a change in pH when the solution is exposed to air
during the retrieval. The eggs are very susceptible to any minute
changes in the pH of their environment. The eggs are washed in this
buffer and then placed into the next medium for culture.

12. 2. FERTILIZATION MEDIUM
• After the wash at retrieval, the eggs are put into the fertilization
medium. This medium contains a variety of salts, sugars, amino acids,
protein and other nutrients essential for the maintenance of the egg
(and sperm in IVF) during the process of fertilization (IVF and ICSI).
The fertilization medium and all of the other subsequent culture
media, are buffered with the appropriate components in order to
maintain the correct pH of the solution in the embryo incubator.

13. 3. CLEAVAGE MEDIUM
• All of the eggs which undergo normal fertilization are next placed into
cleavage medium, which is formulated specifically to support the
growth requirements of the early cleavage stage embryo. The
cleaving (dividing) embryo is cultured in this medium until Day 3. If
the embryo transfer is scheduled for Day 3, the embryos are
transferred to the uterus in a small amount of this medium.

14. 4. BLASTOCYST
MEDIUM
• Embryos, that are to be cultured until Day 5
or 6, are placed, later on Day 3, into another
medium referred to as blastocyst medium.
The embryos are then maintained in this
medium until embryo transfer on Day 5 or
embryo cryopreservation on Day 5 or 6. This
medium has additional components and/or
different components required by the
embryo in its transition from a cleavage
stage embryo to a blastocyst. If the embryo
transfer is scheduled on Day 5, the embryos
are transferred to the uterus in a small
amount of this medium.

15. 5. SPERM BUFFER
• The sperm buffer is formulated in order to maintain the correct pH
when the solution is exposed to air. This buffer is used during the
preparation of semen samples and solutions for semen samples,
which will be washed and processed outside of the incubator.

16. 6. SPERM MEDIUM
• The sperm medium is similar to the Sperm Buffer except that the
buffer is such that the correct pH of the solution is maintained whilst
in the incubator. This medium is important for the final resuspension
of sperm to be used in IVF because the process of fertilization occurs
inside the incubator.

17. HOW LONG SHOULD AN EMBRYO BE
CULTURED
• Two Days: Embryos that are cultured for two days are generally transferred at the
two or four-cell stage. This type of transfer is beneficial for couples who have a
low number of embryos available for transfer, or who have embryos that are
developing poorly.
• Three Days: Embryos that are cultured for three days are usually transferred at
the six to eight cell stage. Many laboratories prefer to culture embryos until this
stage because it allows for increased monitoring.
• Five Days: Embryos that are cultured for five days are transferred at the
blastocyst stage. Blastocysts consist of 12 to 16 cells and are well on their way to
be ready for implantation into the uterus. Many labs opt to transfer at the
blastocyst stage, particularly if you have had repeated miscarriages or IVF failures.

18. MICROMANIPULATION

19. INTRODUCTION
• In 1966, Lin described the technique of micromanipulating and injecting a mouse
egg.
• Subsequently, transgenic animals have been produced by introduction of foreign
genes at the pronuclear stages of fertilized, one-cell zygotes.
• Most of the successes have been with mouse and recently successful production
of transgenic rabbit, pig, sheep and goat have been shown.
• This technique is a powerful tool for studying gene regulation and physiological
functions of gene products in a normal host environment.
• Micromanipulation is the technique whereby sperm, eggs and embryos can be
handled on an inverted microscope stage, performing minute procedures at the
microscopic level via joysticks that hydraulically operate glass microtools.

20. WIDELY USED TECHNIQUES FOR
MICROMANIPULATION
1. Intra cytoplasmic sperm injection (ICSI)
2. Preimplantation genetic diagnosis (PGD)
3. Assisted hatching

21. INTRA CYTOPLASMIC SPERM INJECTION
• The injection of a single sperm into the cytoplasm of the
oocyte, or intracytoplasmic sperm injection (ICSI),
provided a satisfactory solution to the problems of the
assisted fertilization techniques developed earlier.
• In this procedure, a single sperm is first immobilized by
touching the sperm tail with an injection pipette (inner
diameter 5–7 μm). The injection pipette picks up the
immobilized sperm, pierces the ZP and oolemma, and
delivers the sperm inside the oocyte cytoplasm.
• In 1976 using hamsters as a model, Uehara and
Yanagimachi were probably the first to report the injection
of sperm into oocyte cytoplasm (ooplasm).
• It was later attempted on rabbit and human oocytes,
although the first successful human pregnancy was not
reported until 1992 by the Free University of Brussels’
group in Belgium.

22. PREIMPLANTATION GENETIC DIAGNOSIS
What is it?
• Genetic analysis of a single cell from an eight-cell embryo done in conjunction with
in vitro fertilization (IVF) to improve the chances of a “normal” pregnancy.
Why consider PGD in addition to IVF?
1. recurrent miscarriages
2. one child already affected with a genetic disease
3. family history of inherited disease
4. maternal age older than 38
5. prior failure with IVF
6. family “balancing” for sex

23. METHOD OF
PREIMPLANTATION
GENETIC DIAGNOSIS
1. A single cell from the 6-8-cell embryo is removed using a
fine glass needle by puncturing the zona pellucida and
aspirate the cell. - In skilled hands, this generally does not
harm the developing embryo. - Each cell is called a
blastomere.
• Blastomere removal for PGD testing
2. Prepare a metaphase spread of chromosomes to assess
karyotype (number and integrity of each chromosome)
 Two types of assessment techniques are common:
a. chromosome “painting” (or FISH) using fluorescent
probes specific for each chromosome.
b. Polymerase chain reaction (PCR) - amplification of DNA
specific to a gene of interest (family history guides choice
of genes)

24. ASSISTED HATCHING
• Assisted hatching is an IVF technique in which the zona is treated
prior to embryo transfer in order to weaken the wall of the embryo
and thus improve the likelihood of successful hatching and embryo
implantation.
• Just prior to embryo transfer, the developing embryo must "hatch”
out of its outer shell (zona pellucida).
• Some embryos seem to have a thicker shell that may decrease their
ability to implant .
• In such cases Advance technique of IVF with assisted hatching is
performed.

25. TECHNIQUES USED IN
ASSISTED HATCHING
1. MECHANICAL HATCHING : In this the embryo cover is slit
open mechanically with the help of a thin long drawn out
glass needle. This is done with the help of a machine
called micromanipulator
2. CHEMICAL HATCHING : Creating a hole in the zona
pellucida of an embryo by using acidic Tyrode's solution .
3. LASER-ASSISTED HATCHING : this technique involves the
creation of a precise gap in the zona pellucida of selected
embryos using a 1.48 micron infrared diode laser.
Contact laser and Non contact laser
• Contact laser : laser radiation has to be delivered directly to
the zona called contact laser
• Non contact laser : laser gamete manipulation has described
in the non-contact mode.

26. CONCLUSION
• Micromanipulation technology has enabled the reproductive biologist to
overcome inefficient steps in mammalian fertilization, the production of chimeric
animals through blastocyst injection with embryonic stem (ES) cells and the
introduction of specific genes into the genomes of domestic and laboratory
animals.
• This technology has also been used for the production of cloned animals and ES
cell lineages from cloned embryos, using nuclear transfer. Moreover,
micromanipulation is also used for microsurgical embryo biopsy to study the basic
developmental biology of embryos during preimplantation development.