Test Tube Babies - IVF & GIFT
from the book How to Have a Baby:
by Dr. Aniruddha Malpani, MD and Dr. Anjali Malpani, MD.
table of contents
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Egg collection is accomplished today by ultrasound-guided aspiration. This is
a minor surgical procedure that can be done even under intravenous sedation. 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, many doctors
will 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
Insemination, Fertilization, and Embryo Culture
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
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, 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.
About 18 hours after insemination, 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. The 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. 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.
Fig 5. A beautiful 8-cell embryo on Day 3. 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 .
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. 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 on next page]
by Dr. Aniruddha Malpani, MD and Dr. Anjali Malpani, MD.
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