As detailed in Ovulation Induction,
injectable medications including
GnRH agonists, gonadotropins, and HCG will all be administered during
the stimulation cycle to induce a specific response: GnRH agonists to
shut off the secretion of FSH and LH from the pituitary gland to
prevent ill-timed hormonal surges, gonadotropins (primarily FSH) to
control the stimulation of the ovaries to produce eggs, and HCG to
induce the final stages of egg maturation and prepare the follicles to
release the eggs. Also as previously described, a series of
transvaginal ultrasounds to follow follicular development and blood
tests to evaluate proper hormonal response (primarily estrogen) are
used as monitoring tools. These tests are usually performed on day 3 of
the cycle and daily over a 4 to 6 day interval beginning on cycle day
8.
Monitoring allows your
doctor to determine the quality of the cycle so it can be canceled if
stimulation is poor (continuing with such a cycle wastes costly
medications) and, when all goes according to plan, the optimal time for
administering HCG – the final step of the ovarian stimulation process.
Once HCG is given, there’s no turning back. The oocyte retrieval
follows precisely 35 hours later.
Note: Be aware that there
is no single approach to ovulation induction that works equally well
for all patients. Your doctor will be guided by your medical history
and previous responses to induction medications in determining the
stimulation protocol best suited for you.
Oocyte (Egg)
Retrieval
The final stages of
follicular development and egg maturation occur following the
administration of HCG as mentioned above. The scheduling of the egg
retrieval is quite specific at 35 hours after HCG since this is when
the eggs should be at the peak of their development yet just before
ovulation would start occurring. Eggs are removed using the fairly
simple process illustrated at right. The same vaginal ultrasound used
to monitor follicular development during the stimulation cycle is used
here as well, the only difference being the addition of a long, small
diameter needle which runs alongside the ultrasonic probe. The female
anatomy necessitates inserting the needle through the vaginal wall in
order to access the ovaries. Once there, the fluid-filled follicles can
be seen on the ultrasound monitor and aspirated by inserting the needle
into each follicle and engaging the attached suction pump to remove the
fluid. This fluid is collected into a test tube which is examined by a
biologist under a microscope to find and isolate the eggs. Once located
they are quickly transferred to the proper physiological environment
for culture (see Embryology Lab below).
The retrieval is
usually performed with light intravenous sedation and usually lasts 15
to 40 minutes depending on the number of follicles to be aspirated.
Patients typically rest for about 30 to 60 minutes to recover from
the effects of the sedation before being allowed to go home. Your
spouse or a friend will need to be available to drive you home as you
may be a bit groggy for several hours afterward. The vast majority of
our patients tolerate
the procedure very well, the most uncomfortable part being the initial
poke of the needle through the vaginal wall. Like we said, the sedation
is light. Knocking patients out with general anesthesia
brings with it a whole different set of risks and potential
complications that we would rather avoid. As a result, patients may
have some feelings of slight discomfort during the procedure and will
be just conscious enough to be able to listen to commands and respond
to them. The nice thing about the sedation, though, is that the patient
usually won’t remember much about the retrieval once the medicine wears
off. Women may
experience some minor spotting as a result of inserting the needle
through the vaginal wall, but should diminish within a day or two
following the retrieval.
So
we’ve got the eggs, now we need the sperm. Gee, who has the tougher
job? The woman gets to be drugged, poked and prodded with probes and
needles, and requires a day to recover in order to get her eggs. The
man strolls down the hall, goes into a private little room, and
masturbates into a cup. Voila! We have sperm. Hardly seems fair, huh
ladies. See Sperm
Preparation in the
Reproductive Assays Lab section for information on how sperm is
processed for use in IVF.
Embryology Lab
The Embryology Lab
is arguably the most important component in determining the success of
an IVF cycle. Defined culture environments must be maintained here in
order to support egg maturation, fertilization by sperm, and subsequent
embryonic development.
Culture of
Oocytes & Embryos
For 3 to 6 days following
an oocyte retrieval, the eggs, sperm, and subsequent embryos will spend
their time in a biological liquid medium inside a controlled
environment incubator. Maintaining proper culture conditions for IVF
thus requires strict quality control measures to ensure that the medium
and incubator are performing at optimal levels.
The culture medium used in
our lab is commercially prepared and tested for quality by the
manufacturer. This medium is designed to simulate the fluid present in
the fallopian tubes where the eggs and embryos would normally be
developing. Once the medium is received in the lab, it is tested within
our incubator environment using
a mouse embryo test: fresh mouse embryos are collected and placed into
the medium and incubator environments and allowed to develop. The
medium is deemed acceptable for human use if the mouse embryos advance
appropriately.
Now we use what is
called a sequential medium system which basically means that there are
several variations of the medium described above that are introduced to
the eggs and/or embryos as they reach certain stages of development.
For example, we have what we call fertilization
medium as our first medium in the sequence. This
medium is formulated to support egg maturation and sperm survival to
optimize the fertilization process once sperm are added to the eggs.
Once the eggs have been fertilized by a sperm, they will benefit by
being transferred to a slightly different medium composition called growth
medium. Growth
medium is essentially the same core medium with a higher protein concentration and
supports development of the embryos to the 8-cell stage. If we wish to
culture the embryos further, then it is desirable to change the media
formulation once again. This time to a solution called blastocyst
medium. Blastocyst medium has a different chemical composition than the
previous media to specifically support development of the 8-cell embryo
to blastocyst, blastocyst being the most advanced embryonic stage
attainable. Although it is possible to achieve each of these stages of
development in any one of the media formulations described
(that’s what we used to do just a few years ago, in fact), these small media refinements have
allowed us to better match the nutritional requirements of the eggs and
embryos at each phase of growth. As a result, we have observed higher
quality embryo development resulting in higher rates of pregnancy.
Research in the area of culture media refinement continues today,
though, as experts continually strive to develop the "ideal" culture
medium.
Culture medium is
only as good as the environment it is kept in, however, which is why
maintaining precise incubator and lab conditions is equally as
important as testing the efficacy of the medium. Cleanliness of the IVF
lab is priority number one in achieving a successful culture system.
Our lab is treated as if it were a surgical suite by being isolated
from high traffic areas, requiring surgical attire before entering,
being immaculately cleaned and disinfected on a routine basis, using an
air purification system to ensure no airborne contaminants are present,
and when handling eggs and embryos outside of the incubator, using a
laminar flow hood to further ensure exposure to pure air. The
incubators in which the culture system is housed must also be at
peak performance. The incubators are responsible for maintaining
precise temperature, humidity, and carbon dioxide gas levels for pH
control. These components are checked every day by laboratory staff to
ensure they are working properly. Maintaining cleanliness and daily
quality control methods are paramount to achieving a successful embryo
culture system.
Embryology
Embryology refers to
the portion of the IVF treatment where the eggs and resulting embryos
are cultured, handled, and manipulated to achieve a desired outcome by
trained biologists.
Although
these steps were touched on in the previous section, here we shall
provide a little more detail to the processes of oocyte culture,
fertilization, and embryo development.
As
described above, freshly collected oocytes (or eggs) are isolated and
transferred to small tissue culture dishes containing fertilization
medium which are housed in a 370 C, 5% CO2,
humidified incubator. Here, they are allowed to finish the final stages
of maturation and get accustomed to their temporary home.
Note
that not all eggs retrieved will be mature or even normal in
appearance, so you should not expect every recovered egg to result in a
viable embryo for transfer (although it does happen). The quality of
the retrieved eggs is difficult to ascertain at this early stage since
the eggs are actually surrounded by a "complex" of cells making them
difficult to see distinctly. Although they are easily identified as
eggs within these complexes, their level of maturity and quality are
hard to assess at this point. Since most patients ask how the eggs look
at the time of retrieval, we do have some general rules of
thumb to get a rough idea of maturity level, but can’t be
certain of the eggs’ quality until the following day when these cell
complexes will be removed. Now there are techniques of manipulating the
egg complexes in order to visualize maturity and quality of the eggs
within but we, frankly, don’t think it’s that important to know (except
when performing ICSI – see under Micromanipulation below)
and believe the less handling of the eggs outside of the culture
environment the better. The eggs we get will either be mature or
immature, good quality or bad quality and there’s not much we can do to
change them even if we know their status. So we play the hand
we’re dealt – the good eggs should fertilize, the bad eggs probably won’t. And in our
experience, there are usually a lot more good eggs than bad ones.
Insemination
Back
to culturing. After retrieval, the eggs will remain undisturbed in the
culture environment for approximately 6 hours before being exposed to
sperm. The sperm will have been prepared (as described in Sperm Preparation in
the Reproductive Assays Lab section) prior to this time and suspended
in the same medium as the eggs, with the sperm needing some time to
undergo the final stages of their maturation as well, a process called capacitation.
Sperm are added to each dish containing an egg (or eggs) by injection
of a specified volume of the
prepared sperm. This act of adding the sperm to the eggs is called
insemination. We try to inseminate approximately 2 to 3 hundred
thousand motile sperm into each dish. Once inseminated, the dishes are
sealed in the incubator to let the eggs and sperm do their thing.
We
are of the philosophy of adding sperm to each and every egg at the same
time, which supports our contention of not needing to know the specific
quality of each. Years ago, we used to try and add sperm only when we
thought the eggs were mature, with the belief that immature eggs would
be harmed by adding sperm too soon. This meant continually checking on
the eggs, often through the night, and adding sperm when they looked
ready. Often times, there were eggs that never did mature.
This was not only exhausting to the embryologist but contrary to the
tenet of handling the eggs as little as possible. Experience has taught
us that eggs can continue to mature in the laboratory environment, even
in the presence of sperm, and usually only allow a sperm to penetrate
when they are ready to do so. Also, the very immature or poor
quality eggs are not going to fertilize no matter what we do. So why
not inseminate them all at once. Again, the good eggs should fertilize,
the bad eggs won’t.
Fertilization
Check
About
16 to 18 hours after insemination, usually the next morning, the eggs
can be examined for signs of fertilization. This involves viewing the
dishes containing the eggs and sperm under a dissecting
microscope and removing the egg from its cellular complex. In the
presence of sperm, the array of cells, which were initially quite
expansive, sort of dissolve and condense around the egg, completely
obstructing it from view. The embryologist removes these cells by using
a fine glass pipet to gently suction the egg up and down until the
cells flake off and the interior of the egg becomes visible. At this
point, the embryologist looks for the presence of pronuclei within
the egg. Pronuclei present as small circular structures within the
cytoplasm of the egg. Normal fertilization will show 2 pronuclei in
close proximity to each other, with one containing the genetic message
from the egg, the other from a successfully penetrated sperm. These
pronuclei will shortly fuse and disappear so proper timing of the
fertilization check is important. Successfully fertilized eggs are
called zygotes and are transferred to growth medium for further
development. Unfertilized eggs are left in the presence of sperm in the
hope that they still might fertilize, although the potential of doing
so will continue to decline with time.
Embryonic Development
Approximately 24 hours after fertilization has been
established, the embryologist expects to see 2 to 4-cell embryos in
place of the zygotes. These cell divisions are then graded on their
appearance as an estimate of the embryos’ health. High-grade embryos
are those with evenly sized and shaped cells called blastomeres with
a lack of any kind of cellular debris or fragmentation within the
embryo (see pictures at right). Lesser quality embryos are slower to
develop, have uneven or irregular shaped blastomeres and/or presence of
fragments. The more severe the irregularities, the lower it is graded.
Keep in mind, however, that our grading scale should be taken with a
grain of salt, since we are looking only at the external appearance of
an embryo. We cannot see what is going on at the molecular level, so
embryos that may look a bit irregular may be perfectly healthy and
capable of establishing a pregnancy. Similarly, embryos that look
morphologically perfect may be genetically incapable of going on to
make a baby. We see instances of this regularly, so do not be alarmed
if you hear your embryos are not of the highest grade. Grading is more
or less a communication tool for use among the clinical staff to
provide a mental picture of what the embryos are looking like.
After quickly checking for the presence of 2 to 4-cell
embryos, the dishes are returned to the incubator environment for
another 24 hours of culture. So now we’re on the third day following
the egg retrieval and the embryos should be very close or at the 8-cell
stage. Again, the embryos are graded using the evaluation criteria
mentioned above. Depending on the number and estimated quality of the
embryos on this day, the embryologist must decide whether or not to
recommend transferring the embryos back to the patient’s uterus this
same day, or continue to culture them to the blastocyst stage (see day 3 transfer
vs. blastocyst transfer discussion under Embryo Transfer below). If the
decision is made to transfer on day 3 after the retrieval, the patient
is notified and the embryo transfer is scheduled for later that day. If
the embryologist decides to continue culturing the embryos to
blastocyst, the transfer will not be for another two or three days as
this is how long it takes for an 8-cell embryo to develop to a fully
expanded blastocyst (shown at right). Once blastocysts like the one
pictured are obtained, the patient is notified and the transfer will be
scheduled sometime that day.
Micromanipulation Techniques
The term micromanipulation refers to any
procedure where gametes or embryos are physically and individually
worked on using delicate microscopic tools to achieve some particular
end. Here at GYFT, this would involve the processes of intracytoplasmic
sperm injection (ICSI) and assisted hatching.
ICSI
ICSI, or intracytoplasmic sperm injection, is
the process of injecting a single sperm into an egg using microscopic
instruments to do so. It is a relatively new procedure, being widely
used only in the last decade or so, but has revolutionized the
treatment of male factor infertility (refer to Male
Infertility under Diagnosing
Infertility). Prior to its development, men with very low sperm counts,
poor motility, etc. had little chance of fathering their own children,
even with the advancements in conventional IVF available. This was
because the sperm were just too few in number, too weak, or, on the
whole, incapable of penetrating an egg on their own. These men were
simply out of luck since there was nothing more we could do. Who would
have thought that just a few years later, fertilization and normal pregnancies
would be achieved by men with even the poorest sperm quality by
physically placing a single sperm into the center of his
spouse’s eggs. Initially, this procedure met with some skepticism with
concerns that by-passing the normal process of fertilization might
increase the number of abnormal offspring, reasoning that an abnormal
sperm would be expected to create an abnormal baby. This has not
been the case, however. A common analogy is to think of the sperm as a
delivery truck for its DNA payload. Even though the truck may be a
lemon, it doesn’t mean the cargo inside is any less valuable. And
that’s what experience shows us with male factor patients – even though
the sperm are unable to physically deliver their genetic message to an
egg, the DNA is for the most part uncompromised. This is evidenced by
excellent ICSI fertilization rates from even the poorest quality sperm.
Other indications for the use of ICSI involve
past failed fertilization in a conventional IVF setting even though the
sperm and eggs appeared normal and/or oocyte factors such as an
extraordinarily thick or hardened zona possibly impeding sperm penetration
The actual ICSI process is quite labor
intensive and demands great skill and dexterity on the part of the
embryologist in order to achieve superior fertilization rates. At GYFT,
more than 80% of the eggs on which ICSI is performed end up
successfully fertilized, regardless of the nature of the sperm (this
rate is equal to if not slightly better than the fertilization rates
associated with conventional IVF). Keep in mind that the human egg is
roughly the size of a grain of sand with the sperm thousands of times
smaller. It takes a good deal of hand eye coordination and some
sophisticated equipment to pull off this feat. Our embryologists are so
proud of their technique and success that they like to show off by
video recording each ICSI case and reviewing it with the couple upon
completion of the embryo transfer. OK, maybe showing off is not really
their motivation, but they do video tape all of their work to
share with each couple – something we know our patients deeply
appreciate.
Now even though the sperm can be far from
perfect to have a successful ICSI outcome, the eggs cannot. ICSI
requires that the eggs be mature (at the metaphase II stage of meiosis
for you biology majors) in order to have a chance at normal
fertilization. As mentioned in the IVF discussion, it is difficult to
determine the maturity level of newly retrieved eggs because of the
obscuring cellular complex that normally surrounds each. As we also
stated, it’s not that important that we know how mature the eggs are
when performing conventional IVF. With ICSI, however, we must
determine if an egg is mature. Performing ICSI on immature eggs would
be counterproductive and a waste of time in that the egg probably will
not fertilize anyway (or, at best, abnormally fertilize) and you would
prevent the possibility of the egg maturing on its own so that ICSI
could be performed later. Thus, recovery of mature oocytes is critical
to a successful ICSI outcome thereby emphasizing the importance of a
closely monitored ovarian stimulation cycle. Patients should not expect
to produce exclusively mature eggs at the time of retrieval,
particularly when superovulation
may generate as many as 20 to 30 eggs (10 to 12 on average). The goal
is, however, to have most of the recovered eggs be mature –
and this is the case for the vast majority of our patients.
That being said, we go about assessing the
maturity of an oocyte (~ 2-3 hours after the egg retrieval) by placing
the egg/cell complex in medium containing a natural spermatic enzyme
called hyaluronidase which basically works to dissolve the obscuring
cells from around the eggs. Similar to the process of checking eggs for
fertilization (as described above), a fine glass pipet is used to
assist in the complete removal of the cellular complex. At this point,
the egg can now be clearly seen like the one pictured at right.
Maturity is confirmed by the presence of a small bubble-like structure
between the cytoplasm
of the egg and the inner membrane of the egg’s shell, or zona pelucida.
This bubble-like structure is called a polar body. If the egg shows one of
these, then it will be designated for ICSI. Eggs not having a
polar body are returned to the culture environment and checked
periodically over the course of the day for evidence of maturity, still
making it a candidate for ICSI.
Mature eggs are isolated and
returned to the incubator environment to wait while the ICSI equipment
is set up. ICSI is performed using a sophisticated piece of microscopic
equipment called a micromanipulator shown at right. It is essentially a
microscope encased in an incubated acrylic enclosure with the
microtools for performing the technique mounted inside and controlled
from outside the incubated enclosure using severl finely-tuned
joysticks. The microscope is attached to a video camera for recording
the procedures. The microtools used for ICSI are the holding pipet and
the ICSI needle. The holding pipet is used to hold the egg in place
while the ICSI is performed. The ICSI pipet is a very delicate needle
just wide enough to allow a single sperm to enter (actually thinner
than a human hair) and is oriented to enter the egg from the opposite
side of the holding pipet.
Once the microtools have been
installed, several mature eggs are placed in very small media droplets
around the periphery of a petri dish. A single drop of prepared sperm
is placed in the center of the same dish. The drops of eggs and sperm
are kept separated using a layer of mineral oil (the oil also acts as a
protective barrier for the gametes
by helping to insulate them from temperature and pH fluctuations. At
400x magnification, the ICSI needle is lowered into the sperm droplet
where a single sperm is selected, immobilized and picked up with a finely
controlled suction device. The ICSI pipet is raised from the sperm
droplet and lowered with the holding pipet into a drop containing an
egg. Using a small amount of suction, the egg is drawn to the holding
pipet and held in place as the ICSI needle containing the single sperm
is brought toward the egg. The sperm is brought forward to the very tip
of the ICSI needle which is then pushed against and through the zona and into
the oolemma
of the egg. A small amount of ooplasm is aspirated into the needle to
verify complete penetration of the membrane then ejected back along
with the sperm into the ooplasm. The needle is withdrawn and the
suction reversed from the holding pipet to release the egg. The process
is repeated for each remaining mature egg with total time from sperm
selection to completion of the ICSI taking less than 3 minutes per egg.
ICSI’d eggs are then transferred to growth medium in the primary culture
environment until they are checked for signs of fertilization some 18
hours later. From this point on, the steps parallel that of
conventional IVF - from fertilization check to embryo development and
transfer.
Now not all ICSI’d eggs will
fertilize, nor will all even survive. Even though we can be sure a
sperm has been placed in each egg, we cannot guarantee that the sperm
and/or egg will perform as they should and facilitate fertilization.
Fortunately, those that do not fertilize are in the minority, as we
expect a fertilization rate of over 80% as the norm. Occasionally there
will be an egg or two that won’t even survive the ICSI procedure, which
often have to do with the physical nature of the egg. The oolemma of
the eggs can range from very fragile to quite elastic, with the optimum
quality being somewhere in between. Eggs with a very fragile
oolemma are penetrated too easily by the ICSI needle with the
ooplasm often leaking out through the needle entrance which ends up
destroying the egg. Very elastic eggs can be extremely
difficult to penetrate with the needle, and once through, resist the
ejection of the sperm into the ooplasm. These eggs can be damaged from
the trauma of a difficult penetration. The incidence of mortality for
eggs undergoing ICSI is fortunately quite low, however, probably less
than 5%, but does further uphold the importance of superovulation
by making sure we have more than one egg to work with.
Assisted Hatching
(AHA)
The purpose of assisted
hatching is to create a small opening in the zona of an embryo so that it may easily
exit when it has developed sufficiently. This procedure was developed
as a result of findings that egg and embryo zonae can physically harden
or thicken when cultured in
vitro. And despite normal development of the embryo, the
in vitro-induced hardening characteristics of the zona might actually
prevent the transferred embryo from implanting because of a failure to
allow exit of the embryo. Assisted hatching can pretty much guarantee
that an adequate opening will be available for the embryo to pass
through when the time comes. Pregnancy rates here at GYFT rose
significantly since AHA was adopted in 1995. Although some clinics
require certain indications before performing AHA, we have found no
evidence of any harmful effects associated with its use and therefore
decided to perform this procedure on all IVF embryos so that every
couple could share in the benefits that we believe AHA provides.
AHA is almost always performed
on the day of the embryo transfer. The embryologist performs AHA on all
embryos that are expected to be transferred, so if there are more
embryos in culture than we wish to transfer, the embryologist selects
those having the best quality for AHA. After all, we want our patients
to have the best chance at pregnancy which means hatching and
transferring the best-looking embryos. In the event that there are
numerous high-quality embryos available for transfer on day 3, the
embryologist will likely opt to delay transfer until the blastocyst
stage which usually falls on day 5 or 6 after the retrieval (see
day 3 transfer vs. blastocyst transfer discussion below for criteria
and reasons why one transfer may be selected over the other).
The AHA procedure is similar to
that of ICSI (see above) in that it is performed on the
micromanipulator, uses the same holding pipet and the same
microdrop-under-oil setup for isolating the embryos. The difference is
that the ICSI needle is replaced by a hatching pipet, the hatching
pipet being a fair amount larger in diameter than the ICSI needle and
without the sharp, beveled tip. The hatching pipet is loaded with a
slightly acidic solution capable of dissolving the zona quite readily.
With an embryo held in place by the holding pipet, the hatching pipet
is brought near the zona. The acidic solution is then gently ejected
from the hatching pipet, quickly dissolving what it touches until the
hatching pipet is able to just pass through into the perivitelline
space between blastomeres. At the instant of complete penetration,
gentle suction is immediately applied to remove any residual acidic
solution from around the dissolved zona. The hatching pipet is pulled
away and the embryo released from the holding pipet. Embryos are rinsed
further then either returned to growth medium culture to await transfer
that day or moved to dishes containing blastocyst medium to continue
their development.
Embryo
Transfer
Embryo transfer
is the process of placing laboratory cultivated embryos into the uterus
of an appropriate recipient, whether that recipient be the provider of
the eggs herself or a gestational host who agrees to carry the
pregnancy due to some uterine factor associated with the mother. The
biggest question to be answered at this point is how many embryos to
transfer, with the goal to transfer enough to establish a pregnancy but
not so many that we create a high order multiple pregnancy (triplets or
more). And there may be other factors that influence the acceptable
number to transfer such as the patient’s age and/or the quality of the
embryos. Your doctor will provide you with statistics and explain the
risks and benefits of transferring multiple embryos, but ultimately we
wish to do what the individual couple is comfortable with.
Recent
literature on embryo development suggests that, regardless of how good
an 8-cell embryo might look, on average only about 40% of them can be
expected to be genetically normal and capable of reaching a high
quality, fully developed blastocyst. Granted, some patients have better
rates of blastocyst development, others have worse, but the research
maintains that we should expect at least a third to get that far. So by
transferring five 8-cell embryos, we are playing the odds that two of
the five should be capable of developing on to blastocyst in vivo, with
the odds near 50/50 for each blastocyst to implant. So you can see that
a reasonable chance at twins is possible given this logic and, like we
mentioned, we believe twins are an acceptable and safe outcome of IVF.
Depending on the
number and quality of the embryos available on day 3, the embryologist
may suggest waiting for the embryos to develop to blastocyst before
transferring them. If this is the case, we would recommend limiting the
number to transfer to two. That’s because we no longer have to
speculate on the number of day 3 embryos that should be expected to
develop to blastocyst – we already have the blastocysts.
Blastocysts should be expected to implant close to 50% of the time, so
transferring two should give us a good shot at a single pregnancy with
a maximum outcome of twins. Transferring three blastocysts will
increase the chance of a multiple pregnancy, with triplets the maximum
outcome.
Day 3
Transfer Vs. Blastocyst Transfer
Finally an
answer to the burning question, "How do we decide which day to do the
embryo transfer?" There are many different opinions on this subject.
Some clinics only do day 3 transfers, others only blastocyst transfers
because they claim that’s what works best for them. For us, we think we
achieve the best results somewhere in between the two extremes. See if
you can follow our logic:
There
are two major advantages of culturing to and transferring blastocysts.
One is to reduce the risk of high order multiples because fewer embryos
are transferred; the other is to be able to select those embryos that
have shown to be of superior developmental quality for transfer as a
result of reaching the blastocyst stage. So it seems pretty clear that
every patient ought to be transferred at the blastocyst stage, right?
That’s exactly the opinion of some infertility specialists. But what
happens if a patient has only, say, three quality embryos available on
day 3? What is gained by delaying the transfer in this case? We don’t
think anyone would argue with the contention that the laboratory
environment still cannot duplicate all of the benefits of the natural
uterine environment in providing for embryonic development. This being
said, why risk keeping the embryos in the lab and possibly ending up
with no blastocysts to transfer rather than transferring them on day 3
where they will undoubtedly perform better and still have a minimal
risk of multiple pregnancy? The clinics performing exclusively
blastocyst transfers are admittingly willing to have a certain number
of patients not have an embryo transfer.
Then
there are the cases where there are a dozen or more good quality
embryos available for transfer on day 3. The dilemma here is that the
embryologist must try and select a limited number of embryos for
transfer that he/she thinks will develop to blastocyst in vivo. Sure,
it’s easy to pick out embryos that look good but, as stated above, even
the best looking embryos may possess inherent genetic aberrations which
are already predetermined to cease development. According to recent
studies, this will be the case with as many as two thirds of the 8-cell
embryos. So, if a couple has 15 morphologically normal 8-cells on day
3, we can likely expect that only 5 of them have the genetic ability to
achieve blastocyst formation and implant successfully. With no
discernible difference between the embryos, how can the embryologist be
sure he/she didn’t select 6 inherently "bad" embryos for transfer –
odds would be reasonable for this to happen. In this case, it would
make more sense to continue culturing these embryos to actually find
out which ones are capable of developing to blastocyst and transferring
at that time. In this way, we are sure we are transferring the most
genetically competent embryos
Given
these two scenarios, we have developed our own criteria for deciding
when to perform an embryo transfer: If there are six or more
good quality 6 to 8-cell embryos available on day 3, we will continue
the culturing process to blastocyst with the expectation that at least
a third of them will reach that stage. If we have the minimum six, we
should be able to expect the development of at least two blastocysts
for transfer – an ideal number. In cases where we have fewer than six
good quality embryos on day 3, we will transfer that day.
The
actual embryo transfer is a quick, painless procedure nearly identical
to an intrauterine insemination (IUI). The female recipient lies on the
exam table as if she were having a Pap smear performed. The physician
places a speculum in the vagina so that the cervix can be easily seen.
Using an abdominal ultrasound, the assisting nurse locates the uterus
on the monitor. As this is being done, the chosen number of embryos is
removed from the incubator, placed in a dish of embryo transfer medium
and drawn into a long, sterile catheter by the embryologist. This
catheter is then carefully handed to the physician who then inserts it
through the cervix and into the uterus. Once in the uterus, the
catheter can be visualized using the ultrasound monitor, with the goal
to place the embryos near the center of the uterine cavity. When the
catheter is satisfactorily located, the physician injects the contents
of the catheter into the uterus, waits a minute with the expectation
that the embryos will move away from the end of the catheter so as not
to be accidentally drawn back out, then slowly removes the catheter and
hands it back to the embryologist. The embryologist then flushes medium
through the catheter into a petri dish and examines the contents to be
sure no embryos have been retained. On the rare occasion that an embryo
is found, the procedure is
simply performed again. The transfer itself usually takes all of five
minutes, after which the patient is instructed to remain lying down for
one hour to allow the embryos the chance to settle into the uterine
lining. Once the hour is up, a nurse will provide a wheel chair escort
to the entrance where the patient’s spouse will drive her home, keeping
physical activity to a minimum for the next few days to give those
embryos every opportunity to implant. A pregnancy blood test is
performed 15 days after the egg retrieval was performed.
Any
day 3 embryos left in the incubator following the transfer are cultured
further to see if blastocyst development occurs and are frozen if the
patient wishes. Similarly, extra blastocysts present following a
blastocyst transfer are frozen at the request of the patients (see
Blastocyst Cryopreservation below).
Blastocyst Cryopreservation
Cryopreservation is the act of
freezing extra embryos for eventual thaw and transfer, thereby avoiding
the costly ovulation induction step and egg retrieval associated with a
fresh IVF cycle. Historically, embryos have been frozen at all stages
of embryonic development from zygotes to 8-cells to blastocysts. The
current philosophy here at GYFT is to freeze only when expanded
blastocysts are available. This is done for a couple of reasons. One is
that we find that blastocysts tend to survive the freezing and thawing
process better than earlier stage embryos, the reason probably being
that even if a few cells are destroyed when thawing a blastocyst, that
represents just a small portion of the 60 or so cells present. The many
intact cells should be capable of readily replacing the lost cells. In
contrast, if a few cells are destroyed when thawing an 8-cell, this
represents a large proportion of the embryo, making it much more
difficult for the remaining cells to overcome the loss. Another reason
why we only freeze blastocysts is that when you freeze at earlier
stages you are removing embryos from the transfer pool before you know
how all of the embryos are going to turn out. Normally, one could only
justify freezing if there were a fair number of developing embryos
present. In such cases, as we discussed earlier, we would proceed to
blastocyst culture with all embryos so that the superior
quality ones could be identified and transferred, thereby allowing for
the best chance at pregnancy during the current cycle. By freezing
embryos prior to blastocyst formation, you may be removing embryos from
the current cycle that may have had the best opportunity for
implantation or you may be giving false hope to a couple by freezing
genetically unfit embryos that weren’t capable of subsequent
development from the outset. Only by freezing blastocysts do we feel we
have provided the patients with the best opportunity for pregnancy in
the current cycle and cryopreserved only those embryos that have an
excellent opportunity to not only survive the thawing process but have
the ability to resume development and establish a pregnancy as well.
The actual freezing or cryopreservation
process basically involves placing the blastocysts in a freezing
solution composed of glycerol and sucrose, allowing the blastocysts to
equilibrate in the solution, transferring them to small cryovials, then
placing the vials into a programmable freezing chamber where they are
slowly cooled to –400 C then submerged in liquid nitrogen
storage at –1350 C for future thaw and transfer.
Successful embryo cryopreservation requires removal of as
much water from the blastocyst as possible. The presence of water leads
to the formation of intracellular ice crystals which can cause damage
to the embryo when a quickly formed shard of ice slices through vital
membranes. When such embryos are eventually thawed, they are
found to be virtually destroyed. Thus, the importance of glycerol in
the freezing solution. Glycerol is a substance which will freeze
without forming ice crystals, and when blastocysts are placed in a
solution containing glycerol, the water within the blastocyst readily
leaves the embryo in an effort to reach equilibrium with its altered
surroundings. As water leaves, it causes the blastocyst to shrink until
the glycerol slowly diffuses in. Once equilibrium is reached, the
embryo more or less resumes its normal appearance and is ready to be
frozen without less of a risk of ice crystal damage. Sucrose is added
to the freezing solution more to aid in thawing than in freezing. Upon
thawing, the blastocysts must be added back to a water-based medium. If
water is allowed to enter the embryo too fast, it may swell and burst.
The presence of sucrose, however, prevents rapid water inflow by
clogging the pores through which the water normally enters, thereby
slowing down the influx of water to a more comfortable rate. Once the
blastocysts have reached equilibrium with the culture medium, they are
assessed for survivability and placed in the incubator environment to
await transfer (see Embryo Transfer above).
Blastocysts in our program tend to survive the freezing and
thawing process at a 70 - 80% clip, significantly better than when we
froze earlier stage embryos. Thawed, intact blastocysts have shown to
have pregnancy rates close to that of fresh blastocysts with similar
live birth rates.
Luteal Phase Monitoring
After the eggs have been removed from the
ovary (simulating ovulation), it shifts its attention to manufacturing progesterone to
aid in preparing the uterine lining for conception. Since a thick
lining is important for allowing the transferred embryos to implant, we
will want to be sure your progesterone levels are adequate by
supplementing your body with both injectable and vaginal suppository
progesterone. Progesterone supplementation begins immediately following
the retrieval and continues through the embryo transfer until a
pregnancy test is performed 2 weeks later. If pregnant, you will have a
blood test to determine your progesterone level. If adequate, you will
likely be able to continue supplementation using oral tablets. If your
level is borderline or low, you will need to continue the injectable
and suppository supplementation.
Risks of IVF
The risks
associated with the IVF procedure are really no greater than the risks
associated with pregnancy. The minimal risks that are present
are those relating to the ovarian stimulation and the egg retrieval.
The risks associated with ovarian stimulation
have been described in deatil under Risks & Side Effects
of Using Injectable Gonadotropins under the Ovulation Induction section
and deal with irritation and discomfort from the medication injection
sites and the remote possibility of OHSS.
The risks associated with the vaginal
ultrasound-guided egg retrieval are also quite minimal. Potential
problems such as serious internal bleeding caused by the needle are
rarely encountered. The chance of pelvic infection is a possibility but
usually responds to antibiotic therapy should one occur.
The chance of an ectopic pregnancy is still a
possibility even though the embryos are placed into the uterus.
Occasionally a viable embryo will migrate into the uterine end of the
fallopian tube where it ends up implanting and forming a tubal
pregnancy. The incidence of this, however, is very low.
IVF Success Rates
This is probably the only information that
really matters to most patients – what are the chances of getting
pregnant with IVF? Our first warning is to be careful to scrutinize the
answer you get when you ask this question of different IVF programs.
Although all programs are required to report comprehensive,
specifically defined IVF results to the proper regulatory agencies,
it’s really up to the programs themselves how they want to present the
data to prospective patients, and it is in the interest of the program
to present the highest success rate possible. So we suggest you ask
deeper questions like, "Does your program restrict certain patients
from participating?" (i.e. women over a certain age or women having
certain types of infertility). or "Is this pregnancy rate you are
quoting the chance that I will just get pregnant or the chance that I
will actually give birth to a healthy baby?" So use caution when
blindly comparing the pregnancy rates from one clinic to the next.
There are a number of ways to distort success rates by manipulating
statistics or selecting a particular patient population for inclusion
in the data. Similarly, be wary of the program claiming success rates
significantly higher than the norm. Most reputable programs will have
similar rates of success, so when one pops up asserting extraordinary
pregnancy rates, its data deserves a closer look.
The way that we will present our success rates
is by using the standardized method as outlined by SART (Society of
Assisted Reproductive Technologies), which is the statistical record
keeper of ART success rates under the American Society of Reproductive
Medicine. Even SART realizes that certain clinics’ success rates are
not comparable due to the practices mentioned above as they qualify
their statistical summations with the statement "a comparison of clinic
success rates may not be meaningful because patient medical
characteristics and treatment approaches vary from clinic to clinic."
While it is true that every clinic has its own treatment approaches, at
GYFT, we do not exclude patients on the basis of age or type
of infertility from attempting treatment just so we can inflate our
success rates.
Following is a brief explanation of how to
interpret the data in the success rate reports, while clicking the
links at left will let you view recent year reports as published by
SART.
By perusing the success rate reports, you’ll
see that age is the only categorizing variable in determining
the success of an IVF cycle. In all honestly, there are innumerous
variables which could be used to categorize success rates including
type of infertility, severity of infertility, number of embryos
transferred, quality of embryos transferred, number of follicles
produced, preparedness of the uterine lining, ethnicity, weight, and so
on. Because every infertility patient has a unique set of circumstances
pertaining to their difficulty in getting pregnant, it’s quite
impossible to quote a precise probability of success for any
particular couple. As a result, the data is easier to understand if we
group all variables into one large pool of patients when calculating
IVF success rates. We then assess the likelihood of pregnancy for any
given couple based on the single factor of age – a factor which is one
of the more telling in predicting infertility. This all-inclusive data,
then, will include patients with severe infertility issues as well as
those with fairly mild infertility issues. So, depending on the nature
of your particular situation, your chance of success may be better or
worse than the "averaged" rates outlined in the success rate reports.
Looking at the reports, the Program Profile is
the section you will see first. It basically summarizes the
characteristics of our ART services: We are a member of The Society for
Assisted Reproductive Technologies, we offer our ART services to single
women, we support the use of surrogates in ART if needed, and thus far
have not had an instance where donor eggs were shared between
recipients (this is because almost without fail, when a couple goes to
the expense to use donor eggs, they want all the available eggs for
themselves to maximize their chance at pregnancy). In the Type of ART
Used section, you can see that our energies are exclusively devoted to
IVF techniques with about one-third of our IVF cases involving the use
of ICSI (most commonly due to male factor infertility). GIFT and ZIFT
are procedures which, in the past, had better success rates in certain
situations but have since been surpassed in all regards by the current
methods of IVF and are now rarely performed in the U.S. The ART Patient
Diagnosis block simply shows the types of infertility which we
currently treat using IVF.
Now let’s look at the data in the Success
Rates section. The large block of data describes the cycles using fresh
embryos. As mentioned earlier, data is broken down into four age
groups: less than 35, 35 to 37, 38 to 40 and greater than 40.
Number of cycles shows the number of cycle
starts that occurred in each age group during the year (an IVF cycle is
considered started as soon as the first dose of ovulation induction
medication is administered). We’re not what you would consider a high
volume IVF clinic, averaging around 65-70 IVF cycles per year. We
consider this a good thing, however, since it allows our patients more
interaction and attention from our staff throughout the treatment.
Pregnancies per cycle answers the question,
"What are my chances of getting pregnant once I start an IVF cycle?" A
pregnancy is defined as having an initial positive serum pregnancy test
followed by ultrasound-confirmed presence of an intrauterine
gestational sac.
Live births per cycle answers a slightly
different question, "What are my chances of delivering a healthy baby
once I start an IVF cycle?" Notice that this number differs from the
number in the column above due to the fact that not all established
pregnancies as defined above will result in a live birth because of the
unfortunate inevitability that miscarriages do in fact occur. So,
although it can be difficult to assess one patient’s miscarriage rate
over another’s, a woman who believes she is at less risk for
miscarriage may have a live birth rate closer to that of the
pregnancies per cycle rate, while a woman who believes she is at higher
risk for miscarriage may have a live birth rate which is lower.
The live births per retrieval and
per transfer are kept track of because not all patients who
start a cycle get as far as the retrieval and embryo transfer. Thus,
because there are historically more starts than retrievals and more
retrievals than transfers, the live birth rate rises with each
subsequent step of the treatment process that a couple completes. In
recent years, however, the difference between the live births per
retrieval rate and the live births per transfer rate is negligible
since nearly 100% of patients who have a retrieval here at GYFT will
have an embryo transfer as well. Being able to tell couples with
certainty that they will have embryos returned to them following an egg
retrieval bolsters patient confidence in our program because of our
ability to obtain fertilization and embryonic development in
essentially all situations.
There usually is a difference
between the per cycle and per retrieval rates,
however. This is known as the cancellation rate. Cancellations are
cycles which are started but halted at your doctor’s discretion prior
to egg retrieval. Sometimes, once a woman has begun taking her
medications and started being monitored, she may fail to respond
properly or just not respond well enough to justify the expense of
continuing with the current cycle when a better outcome is believed to
be attainable by your physician. Keep in mind that the bulk of the
expense for IVF is in the medications and the processes following and
including the egg retrieval. Thus, it is sometimes in the patient’s
best interest to concede a cycle by having it cancelled prior to the
retrieval and putting those funds toward another attempt should the
clinical staff determine that the current cycle has too poor a
prognosis. Not surprisingly, cancellations tend to occur more
frequently as the age of the woman increases. In younger women
(<35), it’s typical for about 1 in every 20 cycles starts to get
cancelled while women 40 and over tend to have more like a 1 in every 7
to 8 cycle cancellations.
The next line on the success rate report shows
the average number of embryos that were transferred per patient. See
the Embryo Transfer section above for
discussion of the rationale for determining the number of embryos to
transfer.
The final line shows the percentage of
pregnancies resulting in a multiple pregnancy. Historically here at
GYFT, you can expect a twin pregnancy to occur roughly once in every 4
pregnancies and triplets roughly once in every 15 pregnancies.
Multiples tend to be more common the younger the female patient.
The last section entitled "Cycles Using Donor
Eggs" describes the outcomes of all IVF cycles in which donor eggs were
utilized. You’ll notice that most donor egg cycles take place in the 40
and above age group, often because women in this age group are more
likely to be poor responders, have inadequate numbers of eggs in their
ovarian reserves, or have inferior quality eggs in their reserves.
Using donor eggs allows such a woman to significantly improve her odds
of achieving a pregnancy by allowing us to obtain a greater number and
better quality of eggs than she could produce on her own. Incidentally,
donor egg cycles account for most of the pregnancies in the 40 and
above age group.
We hope you are now able to navigate and
comprehend the data in these reports. We also hope you find the
information encouraging as well, as we are quite proud of how we’ve
been able to maintain highly successful results from one
year to the next. It is our mission to constantly employ the latest
technologies and quality control procedures to improve on our success
whenever possible.
