from
the book How to Have a Baby: Overcoming Infertility
by Dr. Aniruddha Malpani, MD and Dr. Anjali Malpani,
MD.
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How
is egg retrieval performed ?
How
is fertilisation checked in the IVF lab ?
How
are embryos cultured in the IVF lab ?
How
is egg retrieval performed ?
Egg collection is accomplished today by ultrasound-guided
aspiration ( www.drmalpani.com/videos/ultrasound-guided-egg-retrieval.wmv).
This is a minor surgical procedure , that can be done
even under intravenous sedation. We prefer doing it
under general anesthesia in our clinic , because we
feel this is safer, kinder for our patients, and allows
us to collect more eggs. The ultrasound probe is inserted
through the vagina. The probe emits high-frequency sound
waves which are translated into images of the pelvic
organs and displayed on a monitor , so that the mature
follicles can be seen as black bubbles on the screen.
The doctor guides a needle through the vagina into each
mature follicle. The follicular fluid containing the
egg is then sucked out through the needle into a test
tube, and all the follicles are aspirated, one by one.
This is a very precise procedure, which requires considerable
skill, and takes about 10-40 minutes to perform, depending
upon the number of eggs. On an average , we retrieve
about 4-16 eggs for each patient. If there are few eggs,
we flush each follicle, to ensure that each egg is retrieved.
The older method of performing egg retrieval
involved a laparoscopy, and the eggs and follicular
fluid were aspirated under direct vision. However, this
method is rarely used today, because the vaginal-ultrasound
guided method is much quicker, easier and safer.
Fig 1. Schematic of egg collection under vaginal ultrasound
guidance. If you click on the picture, you can watch
a video of an actual egg retrieval procedure done in
our clinic
The aspirated follicular fluid is then immediately carried
into the laboratory ( which is adjoining the operation
theater ) where it is examined by the embryologist under
a stereozoom microscope, in order to identify the egg.
Each egg is surrounded by sticky cumulus cells, and
is called an oocyte-cumulus complex. These are washed
in medium, graded for their maturity and then transferred
into the CO2 incubator The maturity of an egg determines
when the sperm will be added to it (insemination). Insemination
can be done immediately upon harvest, but is usually
done after 2-6 hours.
Fig 1. Checking the eggs under the stereozoom microscope
in the IVF lab. If you click on the picture, you can
watch a video of an actual egg retrieval procedure done
in our IVF laboratory
Fig 2. Mature oocyte cumulus complex, as seen under
a stereozoom microscope in the IVF lab, during egg retrieval.
The egg is in the center, surrounded by the cumulus
cells.
On the day the eggs are harvested (
this is called Day 0) , the husband provides a semen
sample. The sperm are separated from the seminal plasma
in a process known as washing the sperm, and these washed sperm are
used to inseminate the eggs. Some men may have considerable
difficulty producing a semen sample at the appropriate
time, because of the tremendous stress they are under,
and the " pressure to perform". For these men, using
a previously stored frozen sample can be helpful. Viagra
( sildenafil citrate) can also be used to help them
to get an erection, as can using a vibrator.
A defined number of sperm ( usually
100,000 sperm/ ml) is placed with each egg in a separate
dish containing IVF culture medium. The dishes are placed
in a CO2 incubator with a controlled temperature that
is the same as the woman's body - 37 C. The conditions
in the incubator and the culture medium are designed
to mimic the conditions in the fallopian tube, so that
the embryos can grow happily in vitro. The culture medium
, which has to be very pure, contains various ingredients
such as protein, salts, buffer and antibiotics which
allow optimal growth of the embryo – think of it as
"chicken soup for the embryo " !
Fig 3. A view of the incubator - the heart of an IVF
lab.
How is fertilisation
checked in the IVF lab ?
About 18 hours after insemination (
this is called Day 1) , the embryologist checks to see
how many eggs have fertilized. This is called a pronuclear
check, and normally fertilized embryos at this time
are single cell , with 2 pronuclei.( Fertilisation
Video ) Each pronucleus appears as a clear bubble
within the embryo, and the male pronucleus represents
the genetic contribution of the husband , while the
female pronucleus represents the contribution of the
wife. When these fuse, a new life, with a unique genetic
composition is formed. Abnormally fertilized embryos
( for example, those with three pronuclei), or those
which have failed to fertilise, are discarded, or used
for research.
Fig 4. A normal 2-PN embryo on Day 1 ( about 18 hours
after egg retrieval) . This is a good quality embryo,
because the two pronuclei ( the clear bubbles in the
center) are touching each other; and the pronucleoli
they contain are aligned properly. If you click on the
picture, you can watch a video of how the sperm fertilise
the egg as seen under the microscope in our IVF laboratory
Fig 5. A beautiful 8-cell embryo on Day 3 ( about 72
hours after egg retrieval) . This is a Grade A embryo,
with regular, equally sized, clear blastomeres; and
no fragments
There is quite a lot of suspense and
anxiety till you find out from the lab how many embryos
have fertilized. This is a biologic variable which we
still cannot control. Sometimes, even though the eggs
and sperm may look excellent , there may be a total
failure of fertilization. This can be a major blow,
because it means that there are no embryos to transfer.
Poor fertilization rates may be because of : poor lab
conditions; a sperm problem, or an egg problem. If only
one patient has poor fertilization on a particular day,
in a good lab, then it’s usually the sperm which are
held to be responsible .
How are embryos cultured
in the IVF lab ?
The normally fertilized embryos are
left in culture, where they continue to divide, and
their quality graded after another 24 hours. Good quality
embryos divide rapidly; and healthy embryos have 2-4
cells, of equal size, with clear cytoplasm and few fragments
on Day 2 ( about 48 hours after egg retrieval) . The
IVF lab is the heart of the IVF clinic today, and an
IVF clinic is only as good as its lab ! Unfortunately,
most patients have no idea of what happens in the lab,
and they rarely get a chance to talk with the embryologist,
the skilled biologist who works in the IVF lab. The
embryologist is the unsung hero of IVF treatment who
does all the important work behind the scenes. The dramatic
improvements in pregnancy rates with IVF today are because
of the important contributions embryologists have made
to finding the best ways of growing and culturing embryos
in vitro.
Many patients are worried that their
eggs, sperms or embryos may get mixed up with someone
else’s. While this can happen, the probability of it
happening in a well-run laboratory is very low, because
good labs have quality control mechanisms to prevent
such mixups from occurring.
After 48 – 72 hours, when embryos usually
consist of two to eight cells each, they are ready to
be placed into the woman's uterus. This procedure is
known as embryo transfer.
continued
. . .
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