|Facts About Embryos
a new sun rises for me; everything lives, everything is animated,
everything seems to speak to me of my passion, everything invites me to
-Anne de Lenclos
Want to learn about embryos? Learn some of the answers to some of the most Frequently Asked Questions here.
How are embryos created during an In Vitro Fertilization cycle?
In Vitro Fertilization (IVF) involves stimulating a woman’s
ovaries to produce more than one egg through the use of fertility
medications. As the eggs are developing, the woman is monitored
through blood tests and ultrasound. When her two largest follicles
reach the optimal size, she is given an injection of hormones to
trigger ovulation. 36 hours later, the eggs are retrieved through
a minor surgical procedure performed under light sedation. Using
ultrasound guidance, the fluid-filled follicles are aspirated with a
needle inserted through the vaginal wall. An embryologist then
examines the collected fluids under a microscope to isolate the
eggs. The eggs are placed into tissue culture dishes with
fertilizing medium and placed into an incubator for several hours prior
to attempting fertilization. Fertilization is achieved by
introducing prepared sperm into the culture dishes, returning them to
the incubator, and allowing nature to take its course. In some
cases, eggs are fertilized manually by injecting an individual sperm
directly into each egg (Inter Cytoplasmic Sperm Injection or
ICSI). The resulting embryos will continue to develop for 3 to 5
days, when they will be transferred to the waiting uterus via a
catheter inserted through the cervix. In most cases, the intended
mother is given hormones to support an optimal uterine lining for
Fertilizing all the mature eggs retrieved allows for the selection of
the best quality embryos for transfer to the intended mother’s
uterus. In order to reduce the possibility of triplets or
higher-order multiples, the number of embryos transferred is usually
limited based on the age of the egg provider and the medical history of
the participants. The remaining embryos may be cryopreserved,
providing additional opportunities for pregnancy without the expense of
an entire IVF cycle. When their family building is complete, some
IVF patients generously choose to donate their remaining cryopreserved
embryos to someone else who needs assistance in order to conceive.
At what stage are embryos frozen?
Embryos may be frozen at any stage of development between 1 and 6 days.
What is an oocyte? Is it the same as a gamete?
An oocyte is an egg. Gamete is a gender-inspecific word that refers to both eggs and sperm.
What is the difference between an embryo and a pre-embryo?
Embryo and pre-embryo are both terms that are used
broadly to refer to all the early stages of development of a fertilized
egg. The word embryo is specifically used to refer to the stage
of development between implantation in the uterus and the 8th week of
gestation, so some IVF clinics use the term pre-embryo when describing
pre-implantation embryos. However, both words are commonly used
to refer to stages of development prior to transfer to the uterus.
What is a 2PN embryo?
Male and female gametes (eggs and sperm) each have a
nucleus containing DNA. Within hours after penetration by the
sperm, the egg jettisons half of its chromosomes in a process called
meiosis. (Sperm complete meiosis much earlier, while still in the
testes.) The remaining DNA packet from each gamete is called a
pro-nucleus. For a period of time after the sperm penetrates the
egg, the two pro-nuclei exist separately within the cytoplasm of the
egg. Appropriately, this earliest stage of development is called
the “two pro-nucleate” (or 2PN) stage. When embryos are
being created in an IVF laboratory, the embryologist typically will be
looking for proof of initial fertilization (2 pronuclei in close
proximity to each other) about 16-18 hours after insemination.
What is a Zygote? What are Blastomeres?
Gradually, the two pro-nuclei gravitate toward each
other, their cell walls dissolve, and they complete the process of
fertilization by merging to form one nucleus. The fertilized egg
is now called a “zygote.” The nucleus begins to duplicate itself
by dividing into cells called blastomeres. This process is called
mitosis. At first, the blastomeres are rather large, but each
division produces smaller and smaller blastomeres. Normally,
embryos reach the 2-4 cell stage about 24 hours after fertilization
(two days after egg retrieval), and a day later, they will have about 8
cells. Here in the United States, Day 3 is the earliest stage at
which embryos are routinely transferred to the uterus. In between
fertilization and the point at which there are too many cells to count,
they are called cleavage-stage embryos. They are also commonly
referred to as 6-cells, 8-cells, etc., according to their stage of
What is a Morula?
When the embryo reaches about 16-32 cells, it is called a morula (mulberry shaped).
What is a Blastocyst?
As the morula continues to divide, it forms an outer
shell of cells (the trophoblast) around a fluid-filled cavity (the
blastocele) containing an inner cell mass (the embryoblast). At
this point, the organism is called a blastocyst. An embryo
usually reaches the blastocyst stage around Day 5, which is when it
would normally be entering the uterus during a natural cycle. The
trophoblast and embryoblast cells will continue to differentiate from
one another as cell division continues within the blastocyst, laying
the groundwork for the placenta to grow from the trophoblast, and the
fetus to develop from the inner cell mass.
How does implantation occur?
Around Day 6, the blastocyst will start to “hatch,”
or break out of the walls of the egg (the zona pellucida), which can no
longer contain the cell division occurring within them. The
trophoblast (outer ring of cells) secretes an enzyme which prepares the
uterine wall for implantation. Trophoblast cells invade and destroy
cells of the uterine lining, creating blood pools to stimulate new
capillary growth. Part of the trophoblast will eventually become
the placenta, and within 13 days after ovulation, chorionic villi
fingers form to firmly attach the developing placenta in place.
What is assisted hatching?
Assisted Hatching is a micromanipulation procedure
in which a tiny hole is made in the membrane surrounding the embryo
(the zona pelllucinda). This membrane holds the blastomeres
together, and during natural conception, it helps prevent premature
implantation in the fallopian tubes. Normally it disappears right
around the time the blastocyst is preparing for implantation in the
uterus. Sometime, due to the age of the oocyte provider, or to
freezing or other factors, the membrane is thick and the growing
blastocyst is unable to break out. In such situations, Assisted
Hatching may be employed. Assisted Hatching utilizes either a
lazer, or a weak acid solution that is precisely applied, then
suctioned away. The embryo is then rinsed to remove any residue
of acid, and returned to the culture dish to be further incubated or
await transfer. There is an increased risk for identical twins
when assisted hatching or other micromanipulation techniques are
Is it better to transfer 8-celled embryos or blastocysts?
There is some difference of opinion about whether it is better
to transfer embryos on Day 3 (cleavage stage), or Day 5
(blastocysts) The answer may be that it depends upon the
protocols of your clinic. Some clinics have not observed a
significant difference in pregnancy rates between the two procedures,
or speculate that embryos which do not survive to the blastocyst stage
in the laboratory might have survived inside the body. However,
the argument for incubating embryos longer in the laboratory is
twofold. One, it delivers embryos to the uterus at the time they
would normally arrive, avoiding exposure to potentially harmful amino
acids and carbohydrates. Improvements in culture mediums may
provide a more hospitable environment in vitro. Two, it allows for the
identification of the strongest, most resilient embryos in the batch,
avoiding the possibility that the most viable embryo(s) might be lost
during the freezing/thawing process rather than selected for the fresh
transfer. Transferring fewer, more robust embryos results in a
lower risk of high-order multiple pregnancies (triplets, quadruplets,
etc). In many clinics, the transfer of blastocysts results in
higher pregnancy rates per transfer, but it also increases the number
of couples who do not have any viable embryos at the time of
transfer. On average, about 30% of embryos make it to the
blastocyst stage, while about 70% stop growing. It is unknown
whether these embryos would have survived if transferred earlier, and
therein lies the debate. (Actual rates vary between clinics and
individual patients. In general, fewer embryos made from the eggs
of older patients, and more embryos made from the eggs of donors and
younger patients, go on to develop into blastocysts.) Often,
clinics will choose extended incubation only when there are sufficient
quantities of good quality embryos on Day 3 (8-10 or more) to assure
the probability of an adequate number of Day 5 blastocysts for transfer.
How are embryos frozen and stored after an IVF cycle?
Very carefully! During the freezing process,
even under the stringent protocols used by IVF labs, embryos must
survive the dangers of ice formation, extreme changes in temperature,
pH and the concentration of electrolyte in their environment, as well
as transmembrane water movement.
The cells of embryos, like other living tissue, contain water.
Water forms sharp ice crystals and expands as it freezes, which would
cause the embryo to burst if it were not removed first. Freezing
also builds up high concentrations of salts in water, which would be
detrimental to the embryos. In order to prevent this problem, the
water is removed from embryos and replaced with cryoprotectant fluid,
which does not form ice crystals. The embryos are briefly
transferred to a phosphate-buffered medium for 5-10 minutes, then mixed
with a highly osmotic cryoprotectant fluid. The embryo
temporarily shrinks as it rapidly releases the water in its cells
through diffusion. Then it swells back up again as cryoprotectant
replaces the water. It only takes a few minutes, and the embryos
are ready for freezing. The exact protocols vary from clinic to
clinic, but follow a similar process.
Embryos are placed in glass or plastic straws for storage.
Typically 1-4 embryos from the same batch are stored in each straw,
based on clinic protocols and how many are expected to be thawed and
transferred together in the future. The straws are pre-cooled,
then placed into canisters that are lowered into temperature-controlled
insulated storage tanks (dewars) of liquid nitrogen. The gradual
freezing process takes several hours.
How long does it take for embryos to thaw?
Embryos thaw to room temperature within less than a
minute or two after being removed from cryostorage, but the entire thaw
process takes about 40 minutes before the embryo is ready for transfer
or further incubation.
How does the thawing process work?
The embryos are removed from their canister and
brought to room temperature. In a reversal of the preparation
process for freezing, the cryoprotectant is gradually removed and
replaced with water by incubating the embryos in increasingly dilute
solutions, each containing more water and less cryoprotectant. Again,
the embryo may burst when water rushes into the cells, so this process
is carefully controlled. Then the embryo is brought to body temperature
and transferred or is placed into culture medium in an incubator until
transfer. Embryos are normally reevaluated before transfer.
Will all the embryos survive the thaw?
How well embryos survive the thaw depends upon the
freezing and thawing protocols used and the quality of the embryos
before freezing. Embryos graded higher before freezing tend to be
more resilient to freezing and thawing. Many clinics will not
freeze embryos unless they are of suitable quality. On Day 3, they
should have at least 6 cells, and no more than moderate (20-25%)
fragmentation, or they are unlikely to survive the procedure.
Typical embryo thaw rates vary from 50-80%, with many labs achieving
>70% routinely. It is important to ask about the thaw rates at
the clinic you will be using.
After thawing, embryos are evaluated. Some
will have no surviving cells. These embryos are called
“atretic.” Others will be partial survivors, with some cell
damage. Large numbers of embryos fall into this category.
The damage may be minor or extensive. Though subsequent pregnancy
rates do have an inversely proportionate relationship with the extent
of damage suffered, these embryos are still capable of resulting in a
live birth, and often recover. This was dramatically demonstrated
with the very first live birth after a frozen embryo transfer, which
occurred in Australia in 1984. The embryo was frozen with 8
cells, but 2 cells were destroyed during freezing/thawing. It was
transferred with 6 cells, survived, implanted, and eventually proved to
the world that successful human embryo cryopreservation was possible.
Since then countless children have been born thanks to this procedure,
often after the transfer of embryos that have experienced some degree
of cell damage.
The very best quality embryos are those that survive with 100% of their
cells intact. It has been suggested that these embryos are
essentially as viable as if they had never been frozen, but fewer
embryos tend to fall into this category.
Embryos with an even number of cells seem to have an edge over embryos
with an odd number of cells; they survive freezing at apx. 5-10% higher
rates. Embryos created with donor eggs seem to freeze better than
embryos of the same grade created with infertility patient’s eggs; apx.
2-5% more of them survive freezing.
Survival rates may also be affected by the stage of development at
which the embryo was frozen. There is some evidence that embryos
frozen at earlier stages of development survive freezing better,
perhaps because they are simpler in structure and still have an intact
nuclear membrane. (For this reason, if there are a very large
number of good quality embryos, a portion may be frozen at the 2PN
stage, and a portion incubated to produce 8-cells or blastocysts for
transfer.) However, it is also postulated that blastocysts may
survive better because they have more cells, and therefore can recoup
easier from the loss of some of them. In practice, this is a
variable that may be lab-dependent, since there are a number of
variations to the protocols used for freezing and thawing. What
is most relevant is what your clinic has found works for them.
How long after they are thawed are embryos transferred?
Blastocysts and embryos frozen at the cleaved stage
can be transferred shortly after thawing (0-6 hours), but embryos
frozen earlier on are frequently incubated for a day and transferred
when they reach the cleaved or blastocyst stage.
How long can embryos be frozen and still be viable?
Theoretically, embryos may be frozen indefinitely,
as no biological activity takes place during cryopreservation. In
practice, it is unknown how long they can remain viable. The
first frozen embryo transfers resulting in live birth took place in the
mid-eighties. Since then, embryos frozen as long as 12 years have
resulted in successful pregnancy. Freezing and thawing protocols
continue to improve, making it possible that embryos frozen more
recently may have better outcomes.
Will frozen embryos be as likely to produce a pregnancy as fresh embryos after they are thawed?
The success rates for frozen embryo transfers are
usually lower than those for fresh embryo transfers. This may be
due to damage to the embryos during freezing, but may also be due to
the fact that the embryos evaluated to be of the highest quality in the
batch are usually transferred fresh. In addition, more patients
undergoing a frozen embryo transfer have had a failed cycle than those
undergoing a fresh transfer.
Can embryos be frozen more than once?
Yes. Sometimes when a large quantity of
embryos are frozen at an early stage of development (2PN), all or a
good number of them will be thawed and incubated. As with a fresh
transfer, the best quality embryos will be selected for transfer and
the remainders re-frozen. Live births have resulted from the
transfer of twice-frozen embryos, but some of the same factors that
make frozen embryos transfers less effective than fresh transfers would
be expected to reduce the success rates for refrozen embryos.
What are the factors thought to affect embryo quality?
Age of egg provider
Method of ovarian stimulation
Timing of ovulation trigger
Length of ovarian resting time since last IVF cycle
Quality of Sperm (DNA fragmentation, etc)
Embryology Laboratory protocols and expertise (including culturing environment)
Polycyctic Ovarian Syndrome
What is fragmentation?
Embryo fragmentation occurs when cells divide unevenly, creating bits
of membrane-bound cytoplasm that have no nucleus. The majority of
embryos created during IVF cycles are observed to have some
fragmentation, however, the more fragmentation, the less likely an
embryo is to develop normally and implant successfully. The
extent of fragmentation is an important indicator of embryo
quality. Some research has also shown that the size of fragments
also has an effect on embryo viability.
How are embryos graded?
Different clinics use different grading systems, so
it is important to ask what system is used at the clinic where the
embryos were created. Either a numerical or alphabetical grade is
given based on the rate of cleavage and overall quality. A 3, 4
or 5-point scale may be used. Quality is most often evaluated
based on appearance, but a few labs are now using a more sophisticated
chemical marker test (sHLA-G) to identify the embryos with the best
potential for implantation. Generally speaking, a better quality
embryo is more likely to survive freezing than a poor quality embryo
created on the same day.
In a typical scheme in which embryos are graded from 1 to 4 (with Grade
1 being the best), Grade 1 embryos are morphologically perfect, with
round, symmetrical cells. They have the appropriate number of
cells for their stage of development. Cell division is even, and
there is no visible fragmentation. Only 20% of embryos receive
the highest grade. Most embryos have slight imperfections, and
this is normal and expected. Grade 2 embryos have the correct
number of cells for their developmental stage, and usually have even
cell division. They might have slight unevenness of blastomere
sizes and/or a small amount of fragmentation (<20%). Grade 3
embryos have uneven cell division and moderate fragmentation (>30%),
or are behind in their development, or have similar irregularities.
Grade 4 embryos have uneven cell division and extensive fragmentation
(>50%). They may have few viable cells or be completely
fragmented. Generally, embryos graded at the bottom of the scale are
associated with poor chances for a viable pregnancy. Keep
in mind that this is just an example of a grading system. The
actual parameters used to evaluate embryos vary somewhat and grading is
At some clinics, blastocysts are graded with a number and two
letters. The number refers to the amount of expansion, the first
letter to the quality of the inner cell mass that will develop into a
baby, and the second letter to the quality of the outer cell mass that
will become the placenta. Again, it is best to check with the
clinic where the embryos are created for an explanation of the grading
What is “normal development” for an embryo at the time of transfer?
Hours after insemination or ICSI:
4 cells by 48 hours
8 cells by 72 hours (3 days)
Morula stage by 96 hours
Blastocyst formation by 120 hours (5 days)
What is PGD?
Preimplantation Genetic Diagnosis (PGD) involves
microsurgical removal of one or two blastomeres from a 6 or 8 cell
embryo (generally on day 3). Removal of a cell at this point in time
does not appear to harm the embryo. Various technologies are then
employed to test the embryo for chromosomal abnormalities. When
appropriate, cells from the egg’s polar body may be tested instead and
these cells can be removed much earlier. The polar body, which normally
dissolves at implantation, does not have any known role after
fertilization, so this is considered less invasive.
PGD can be used to detect chromosomal abnormalities or problems with
genetic coding that will lead to later miscarriage or heritable
diseases. Allowing embryos to develop to the blastocyst stage before
transfer weeds out most monosomies (embryos with one, rather than two,
copies of a chromosome). These embryos tend to cease developing early
on, around day 4. Trisomies (embryos with three copies of a
chromosome, as in Down’s syndrome) account for about one forth of all
spontaneous abortions. They may look just like other embryos, so
PGD is used to identify them.
PGD-FISH (fluorescent in situ hybridization) can detect extra, missing
or translocated chromosomes. PGD-PCR (polymerase chain reaction)
can identify specific mutations such as those causing Cystic Fibrosis,
Tay-Sachs disease, sickle cell anemia, muscular dystrophy, and
X-chromosome linked disorders. When PGD-PCR is used, the genetic
code in the selected blastomere is duplicated many times over so that
it is possible to detect the genetic code that would result in a
Testing usually takes 24-48 hours, after which embryos can be selected
for transfer based on the information gained. PGD is not
foolproof, because some embryos contain both normal and abnormal cells
(a condition called mosaicism) and the particular cell selected for
testing may not be representative. But it is a very useful tool,
with accuracy between 90-98%. Where there are no chromosomally
normal embryos, the chances of a live birth are low. This happens
most often when the egg provider is over 40. PGD can increase the
cost of a cycle by $2,500 and $5,000.
Is it possible to have genetic tests performed on embryos that have been frozen?
Yes, PGD may be performed after the embryos are thawed.
Is it possible to tell the gender of an embryo?
Yes. PGD (see above) can also be used to
determine sex based on chromosomes (XX for females; XY for
males). Gender selection may be used to avoid certain genetically
transmitted diseases that are only carried by male or female
offspring. The Ethics Committee of the American Society of
Reproductive Medicine (ASRM) discourages the use of PGD to select
gender for non-medical reasons. Couples who are seeking to
balance their families with a child of a specific gender may also use
techniques like sperm sorting (i.e., MicroSort) to increase their odds
of the hoped-for outcome.
What is the typical success rate with frozen embryo transfer?
The Centers for Disease Control and Prevention (CDC)
publishes an annual report on Assisted Reproductive Technology (ART)
statistics in the United States. The information is self-reported
by participating fertility clinics. The most recently published report,
from 2002, indicates that the live birth rate for transfer of frozen
embryos created with non-donor eggs is 28%, and when the embryos were
created with donor eggs it is 38%. The report does not contain
information on the success rates for donated frozen embryos, but it
would be reasonable to expect that similar success rates would apply,
depending on whether the embryos were originally created with donor
eggs or an IVF patient’s eggs.
When do embryos implant in the uterus?
During a natural cycle resulting in conception, an
egg released from the ovary is fertilized by sperm in the fallopian
tube, and arrives at the uterus 3 to 5 days later. If it is still at
the morula state when it enters the uterus, it will take a day or two
to develop into a blastocyst. Either way, implantation normally
occurs 5-8 days after fertilization, with day 6 being the most common
day. During IVF cycles, effort is taken to simulate the natural
state of affairs, so that the uterine lining is in synch with the
development of the embryo and receptive to implantation. Frozen
embryos are usually transferred on Day 17 or 19 of a menstrual cycle
(depending on the developmental stage of the embryos).
What are the symptoms of implantation?
Some women experience light bleeding (spotting) or
cramping at the time of implantation, but others feel perfectly
normal. The only way to initially tell if implantation has been
successful is to take a pregnancy test.
How soon can I take a pregnancy test?
Pregnancy tests measure human chorionic gonadotropin
(hCG) which is produced by the placenta during pregnancy. Home
pregnancy kits test for hCG in urine; professional beta serum pregnancy
testing measures hCG in the blood. The sensitivity of home
pregnancy tests varies from as low as 15 mIU/mL to 100 mIU/mL or
higher, whereas some beta tests can detect hCG in the blood as low as 5
mIU/mL, which can occur within a week of embryo transfer. (<5%
is considered negative; >25% is considered positive) 85%
of the time, in the early stages of a normal pregnancy, hCG levels
double about every 48-72 hours. Since many doctors require their
patients to wait two weeks after their transfer before administering a
beta blood test, a sensitive home pregnancy test can often be used for
first confirmation of pregnancy.
What factors can be used to assess the likelihood that an FET will be successful?
The three most important factors affecting FET pregnancy rates have been shown to be:
Embryo grade after thaw
Number of embryos transferred
Number of embryos transferred with 100% cell survival after thaw
Factors that may come into play include:
Age of the ovum provider at the time of egg retrieval
Sperm quality (factors like DNA fragmentation begin to affect development after Day 3)
Embryo grade and cell division prior to freezing (affects survival)
Stage of development at time of transfer (slightly higher rates of implantation for blastocysts)
Whether assisted hatching was performed (some evidence this improves implantation rates for previously frozen embryos)
Uterine environment (bloodflow, miomas, uterine birth defects,
malfunction of the cervix, endometriosis, immune issues,
infections and various other factor may affect implantation)
Whether a live birth resulted from the fresh transfer for the same batch of embryos.
Are children born from frozen embryo transfer normal?
The cryopreservation process does not appear to
adversely affect birthweight, perinatal mortality rates, birth defects,
growth or health of children during infancy and early childhood over
children born from fresh IVF transfers. However, the long term
affects of cryopreservation over a lifetime have not been studied
extensively in humans, and since this technology is only about twenty
years old, most of the children whose parents utilized cryopreservation
are still in the process of growing up.
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report. A. Revel, A. Safran, N. Laufer, B.E. Reubinov and A.
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The Miracles Waiting, Inc. Team
Information provided in this page is for educational purposes only and
you should not rely on it as a substitute for professional assistance.
If you are concerned about your health or the health of a loved one
please seek the help of a medical care provider.
|Nearly 400,000 embryos are stored in the United States,|
88.2% are targeted for patient use, and
2.8% are available for research.
FERTILITY AND STERILITY