Patients who have been undergoing ovarian
stimulation for in vitro fertilization (IVF) must take daily injections of
hormones to stimulate the growth of follicles in the ovaries. Within these follicles are eggs (oocytes)
that must then be retrieved so that they can be placed in a laboratory for
fertilization by sperm. The primary
reason that IVF is so successful is not only through the development of
multiple embryos, but by selecting the best embryos from this group for
transfer into the uterus. In some ways,
IVF is like cramming many months of trying to conceive into one cycle, because
of the many embryos that result, but it enhances the probability of success by
choosing the best embryo(s) from the group to be replaced. In a natural cycle, typically only one oocyte
develops and is ovulated, regardless of the quality.
Advances in embryo culture have made increasing
duration of embryo culture possible. In
the early days of IVF, embryo transfer might have been performed after
fertilization (day 1) or on day 2 after oocyte retrieval. Technical expertise and research studies have
refined optimal culture conditions so that embryos are now routinely cultured
to day 3 or day 5 after oocyte retrieval, and some embryos are even transferred
on day 6. Ultimately, there are
limitations to embryo culture in the laboratory, since the development cycle of
an embryo requires that it hatch from its shell and implant in a receptive endometrium
or fail to develop further.
In order to select the best embryo for transfer, an
embryologist observes embryo development in the laboratory over the course of
several days. On the day following
oocyte retrieval, the embryos are assessed for normal fertilization. Problems in development can arise even this
early fertilization is a chemical reaction that is initiated after a sperm cell
penetrates an oocyte; however, the insertion of a sperm into an egg does not
guarantee that fertilization will occur properly. In addition, sometimes the defense mechanism
of the egg, which should prevent more than one sperm from entering, fails, and
the resulting embryo will be abnormal.
Factors that determine when to transfer embryos
include the age of the patient, the number of embryos that are available to
transfer to the uterus, the quality of the embryos in the laboratory, past
history of IVF treatment and the outcome of those treatments. Determining the
number of embryos to be transferred follows similar considerations, and
guidelines for the number of embryos to transfer have been developed.
In order to choose which embryo(s) to transfer,
several systems to assess the quality of the embryos have been developed, so
that the best embryo can be selected. In
short, the system created to selecting embryos for transfer must be better than
picking embryos randomly. The most
common method for choosing embryos is visual morphologic assessment using a
microscope, an embryologist observes and takes notes on how the embryo appears
using standard criteria. By assessing
embryos daily, the embryologist creates a record by which to compare one embryo
against another so that they may rank in order of choice for transfer. Since embryos are cultured individually in
labeled droplets in a culture dish, a record for each embryo can be created
over the course of days. There are
numerous standardized grading systems for evaluating embryos, but each
laboratory may also set its own criteria for what indicates better quality
embryos based on observed outcomes and may follow unique grading systems.
When evaluating embryos under a microscope,
morphological items of interest may include the symmetry of the cells, the
evenness of cell size, the number of cells, the number of nuclei in each cell,
the amount of fragmentation of the cell, the quality of the shell (Zona
Pellucida), and the clarity of the cell’s cytoplasm. For an embryologist, learning to identify and
judge these characteristics requires extensive training, and expertise in morphological
assessment is gained over time, so it is not surprising when patients don’t
even understand some of the terms reproductive endocrinologists sometimes use
when talking about embryo quality.
In general, we do our best to convey to patients
meaningful information about the quality of their embryos prior to
transfer. Since most people don’t have
an education in human embryology, it can be difficult to convey this
information in a satisfactory way. The ideal embryo to transfer on day 3 after
oocyte retrieval has eight cells.
Sometimes other embryos may have more or fewer cells. Most people seem to understand that slow
development is likely to decrease the probability of success, but not as many
understand that faster development (having more than 8 cells on day 3) may not
actually be better. We suspect that
8-cell embryos are generally the best (excluding other considerations) because
the resulting pregnancy rates seem to be the highest when 8-cell embryos are
transferred. This observation does not
have an easy explanation, but is just something that has been observed. However, it also does not guarantee success,
and it does not always indicate that 8-cell embryos will the best embryos of
any group of embryos available for transfer, just that the probability of
success may be highest when this specific situation is observed.
When other factors are considered, such as the
other morphologic characteristics listed above, other embryos may be found to
be superior in implantation potential.
There are theories as to why the 8-cell embryo is likely to be among the
best embryos of any group: there are “checkpoints” in cell development, much
like a turnstile – the checkpoint that prevents cell division likely does so
because proper conditions for cell division have not been met, possibly because
the cell is abnormal, and checkpoints that allow too much progress (more rapid
cell division) might not be conducting proper quality control and have failed
to slow down abnormal cells or premature cell division.
When there more good quality embryos on day 3 than
are appropriate for transfer and when the quality of these embryos is so
similar that it is difficult to choose which among them are the very best,
embryos are generally cultured for two more days. Typically, embryo transfer is not performed
on day 4 because the stage of development on day 4 is difficult to assess using
a microscope: on day 4, embryos are typically at the “morula” stage, which is
amorphous and lacks clear characteristics by which to judge development.
On day 5, embryos have ideally become
“blastocysts.” Blastocysts have two cell
types: an outer ring of cells that are destined to become the placenta and an
“inner cell mass” that eventually becomes the developing fetus; because
blastocysts typically have more than eighty cells, it becomes impossible to
look at each individual cell to assess it, as is done on day 3. Instead, a quality “staging” system is
typically used. A number score (1-6) to
indicate the degree of expansion, like a shoe size, is assigned, and letter
grades, A-D, like in school, are given, first for the inner cell mass and then
for the outer cells. So, a high quality
blastocyst might be stage: 4AA or 5AB, for example. A stage 6 blastocyst has “hatched” from the
shell surrounding the embryo. Since two
different systems are used, it is impossible to compare day 3 and day 5
embryos.
Day 5 embryos are given letter grades to assess the
quality of the two cell types based on morphologic assessment. For example, numerous cells in a tight
cluster are representative of a better quality inner cell mass than sparse
cells that are loosely associated. On
day 3, letter grades are not typically given, which is often frustrating for
patients. Although we could assign
letter grades to these embryos, it wouldn’t mean the same as on day 5, since
different things are being assessed.
Also, assigning letter grades on day 3 might obscure the real quality of
the embryo: for example, if two students take a math test and one scores a 96%
and the other a 99%, but the teacher gives them both an “A” grade, are they
really the same? By grouping assessments
into more encompassing, simplistic categories, important information is lost. A patient can always ask the doctor to make
such a comparison for ease of mind, but that assessment may obscure the true
quality of an embryo.
In the absence of more specific information, such
as the genetic make-up of a cell, which usually requires invasive procedures
such as embryo biopsy, morphologic evaluation is the mainstay of embryo
assessment. Other methods of assessment,
such as analyzing the culture medium in which the embryo has been growing to
detect markers of embryo health, are being developed, but have not been shown
to be more accurate in determining which embryos have the highest implantation
potential. Not all embryos, even when
morphologically perfect, may be genetically normal, just as not all poor
quality embryos are genetically abnormal, so selecting embryos based on
morphology is not a perfect system. In
addition, it is possible that all embryos are abnormal and no pregnancy will
result or that other factors unrelated to the embryos will interfere with
successful pregnancy despite morphologic assessment. However, selecting embryos based on
morphologic criteria currently remains the best method to choose embryos
without using more invasive techniques.
