Embryo culture involves growing embryos outside the body in an artificial environment before transferring them to the uterus. Embryos can be cultured for 2-5 days in specialized incubators that carefully control temperature, pH, gas levels and humidity. Extended culture to the blastocyst stage at 5 days allows more selection of healthy embryos and mimics natural implantation timing. A variety of culture media are used optimized for fertilization, early cleavage and blastocyst development stages.

1. EMBRYO
CULTURE

2. What is en embryo culture
• Embryo culture is a component of in
vitro fertilisation where in resultant embryos
are allowed to grow for some time in an
artificial medium before being inserted into
the uterus.
•

3. • Embryo culture can be performed in two ways
Artificial medium
autologous endometrial coculture
• Artificial culture medium can be
single culture
sequential culture

4.  Single or sequential medium are equally
effective for the culture of human embryos to
the blastocyst stage.
 Artificial embryo culture media contains-
glucose, pyruvate, and energy-providing
components
 Amino acids, nucleotides, vitamins, and
cholesterol improve the performance of
embryonic growth and development

5. SINGLE CULTURE
• the same culture medium throughout the
period
SEQUENTIAL
CULTURE
• the embryo is sequentially placed in different
media

6. 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

9. The potential candidate
• Coculture can be an effective treatment for
patients who have failed previous IVF cycles or
who have poor embryo quality.

10. EGG RETRIEVING
• Egg is

11. TECHNIQUES IN EMBRYO
CULTURE

12. 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.

13. 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.

14. 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.

15. 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.

16. 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.

17. 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.

18. BLASTOCYST CULTURE

19. BLASTOCYS
T
An embryo that has developed for
five to seven days after fertilization
and has 2 distinct cell types and a
central cavity filled with fluid
(blastocoel cavity)
The cells in a blastocyst have just
started to differentiate
The surface cells that surround the
cavity (just under the outer shell) are
called the trophectoderm and will
later develop into the placenta
A more centrally located group of
cells - the inner cell mass, will
become the fetus

20. Blastocyst development
• The blastocyst usually forms on day 5 as fluid builds
within the compacted morula
• A healthy blastocyst often begins hatching from its
outer shell, called the zona pellucida between day 5 to
day 7 after fertilization
• Within 24 hours after hatching, embryo
implantation after IVF (or a "natural" pregnancy)
begins as the embryo invades into the uterine lining
• The blastocyst releases HCG hormone (the pregnancy
test hormone) which leaks into the mother's blood as
the embryo implants

21. Blastocyst formation rate
• The goal of in vitro fertilization and embryo culture is
to provide high quality embryos which are capable of
continued development and result in live births
• However, under standard IVF culture conditions, only
about 25 to 60% of human embryos progress to the
blastocyst stage after 5 days of culture
• The low rate of embryo development has 2 main
causes:
– A less than optimal culture environment in the lab dish
– The inherent "weakness" of human embryos
• Therefore, in the past embryos were transferred to the
uterus after 2 or 3 days

22. Advantages of blastocyst transfer for
IVF
• One problem with this is that 2 to 3-day-old embryos are
normally n the fallopian tubes, not in the uterus. The
embryo gets to the uterus about 80 hours after ovulation.
• Embryo implantation process begins about 3 days later -
after blastocyst formation and hatching out of the
embryonic shell have occurred.
• Therefore, if in vitro culture conditions are maximized so
healthy blastocysts form at a high rate, then day 5
blastocyst embryo transfer can be done.
– The uterine lining on day 5 should be receptive to the arriving
embryo - this a more "natural" time for the embryos to be in the
uterus. It is the same timing as with a natural pregnancy.
– The transfer is done shortly before the time for actual invasion
and implantation

23. How Long Should an
Embryo Be Cultured?

24. • 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.

25. 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. Embryos
cultured for three days can
be checked by the
embryologist for gene
activation and cleavage,
which improves the
likelihood of transferring a
viable embryo.

26. 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.

27. The Embryo Culture Equipment

28. The Laminar Flow Hood
• The preparation of all media and solutions to
be used in IVF, ICSI and IUI occurs inside this
specialized hood, which blows air out towards
the embryologist. The air is filtered and the
outflow of air prevents any contaminants from
blowing in and contaminating the solutions
and embryo dishes being prepared.
Preparation of semen samples to be used in
IVF, ICSI and IUI also occurs in this sterile
environment.

29. The Preparation Incubator
• All dishes and solutions to be used for an IVF,
ICSI or IUI treatment are maintained in this
incubator until use. The incubator is sterile
inside, is at 37°C, has a carbon dioxide
concentration of 6.0%, and the environment is
fully humidified to prevent any evaporation.
All solutions and dishes to be used for
treatment are equilibrated in this incubator
for a minimum of 4 hours before use.

30. Embryo Culture Incubator
• All eggs and embryos are incubated here
throughout their time in the VFC laboratory. The
unit is infused with the proper levels of oxygen
and carbon dioxide to ensure that the
eggs/embryos are maintained under optimum
conditions at all times. The environment in the
incubator is also humidified and kept at 37°C. The
temperature and gas levels are monitored
continuously and the incubator is attached to a
telephone based alarm system which will call out
to the embryologist during off hours should an
unsuitable or emergency condition arise.

31. IVF Chamber
• Whenever the eggs and embryos need to be
outside of the incubator for any reason, they
are handled in our IVF Chamber. The chamber
looks like an isolate that you would see in a
special care newborn nursery in the hospital.
This chamber however is specially modified
and adapted for the purpose of maintaining
eggs and embryos under optimum conditions
even when they are being handled outside of
the incubator.