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. Regardless
of the day of embryo transfer, a micromanipulation process called assisted hatching will be performed on day 3 following the egg retrieval. Continue reading for a description of this procedure.
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 five years 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. Over the years, we’ve arrived at the number
six as an appropriate number of day 3 embryos to transfer or three as the desired number of expanded blastocysts to transfer
on day 6. These numbers have been borne out by our statistics as well: five years ago, four was the number of day 2 or 3 embryos
that most clinics were transferring and which we were having decent success with. As the years went on, we began tranferring
five day 3 embryos after which we saw a significant increase in our pregnancy rates but a negligible increase in the rate
of multiple pregnancies. Slowly the number drifted up to six day 3 embryos as a routine number for transfer. Again, the pregnancy
rate rose, but this time so did the number of multiple pregnancies, the vast majority of these being twins. Since most patients
are thrilled at the prospect of twins and most women seem to handle twin pregnancies quite well, we considered the rise in
the twin rate to be acceptable to continue transferring six day 3 embryos. Now you may come across other opinions stating
six is too high a number to transfer because of the risk of multiples, but recent literature and our statistics don’t
bear this out. Of course, if anyone is of the opinion that twins are an unacceptable risk, then we’d have to
agree. We do not share such an opinion, however.
Recent literature
on embryo development suggests that, regardless of how good an 8-cell embryo might look, on average only about a third of
them are genetically capable of reaching the fully developed blastocyst stage. Granted, some patients have better rates of
blastocyst development, others have worse, but the research maintains that we should expect a third to get that far. So by
transferring six 8-cell embryos, we are playing the odds that two of the six will 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.
Also, our statistics
do not reflect a significant risk of high order multiples when transferring six day 3 embryos.
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 or three. 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’t
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 development
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 six 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. We don’t run into this dilemma when there
are six or fewer total embryos available since there’s no selection process necessary – we simply transfer everything
we have.
Given
these two scenarios, we have developed our own criteria for deciding when to perform an embryo transfer: If there are eight
or more decent 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 eight, we should be able to expect
the development of two to three blastocysts for transfer – an ideal number. In cases where we have fewer than eight
decent quality embryos on day 3, we will transfer that day. Say we have the maximum seven embryos. Even if all of the embryos
look the same, the embryologist is to select six for transfer. Using the general assumption that two thirds of the embryos
are genetically unfit for establishing a pregnancy, odds are pretty safe that the "good" embryos will be included in the transfer
group.
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.
