The egg when examined at the time of IVF oocyte retrieval is encased in a mass of granulosa cells

called the cumulus complex. These granulosa cells possess the receptors for FSH, and the
gonadotropin medicine taken during the stimulation protocol (i.e. Gonal-f, Follistim, Menopur,
Repronex) act on these cells to stimulate their growth and estrogen production. It is their estrogen that
we measure during the stimulation to monitor the stimulation. The granulosa cells communicate with
the egg as the egg develops and matures. The picture below shows the egg as it is removed from the
follicle at retrieval. Because of all the granulosa cells surrounding the egg, it is difficult to get as clear
picture.

Figure 1

When we do ICSI, we strip the granulosa cells so that we can identify the mature oocyte. The mature
oocyte has a single polar body as shown below. This egg came from the complex in the photo above.

Figure 2

Fertilization check at the pronuclear stage

Approximately 16-18 hours after the eggs and sperm are placed together we make an assessment of
fertilization. The fertilized egg has two pronuclei representing the chromosomes from the male and
female and two polar bodies, the chromosomes that were extruded after the completion of meiosis II.
The picture below shows an unfertilized egg.

Figure 3
Not all eggs will fertilize. You can see there are no polar bodies or pronuclei. Eggs may be immature
or even post-mature and have a decreased ability to fertilize. Luckily, we often have enough eggs that
almost all of the patients will ultimately get an embryo transfer. The picture below shows a fertilized
egg after insemination.

Figure 4

The two pronuclei and polar bodies are clearly seen. The picture of the fertilized egg is not under high
magnification. If we examine the egg at even higher magnification we can see several other
components that may play a role in the potential to produce a viable embryo; orientation of the
pronuclei relative to the polar bodies, pronuclear shape, timing of the nuclear membrane breakdown,
and the presence of nucleolar precursor bodies (NPB). For instance, investigators have shown that
the implantation rates were highest if the two pronuclei did not differ in NPB number by more than 3,
and that the NPB were either both polarized or un-polarized in both pronuclei. Some investigators
have used a special microscope to visualize the meiotic spindle apparatus, the scaffolding system
upon which the chromosome split and suggested that they can pick out the best embryos, however
this has not been confirmed in vigorous trials.

Occasionally we will see three pronuclei in the fertilized egg, suggesting that the egg may have been
fertilized by more than one sperm. This egg is chromosomally abnormal and should be discarded.
You can see the three pronuclei in the picture below.

Figure 5

Assessment of the cleavage stage embryos

Generally we perform day# 3 embryo transfer in our IVF lab. At times we do day # 5 (blastocyst)
transfers depending upon the specific situation. When we assess the embryos on day # 2 and day #3
we are attempting to grade the embryos based on several features seen with the microscope.
Embryos that may have higher potential to implant and produce a viable pregnancy often have
several features; lack of multinucleation of the blastomeres, 4-5 blastomeres on day # 2 and >6
blastomeres on day #3. It is of note that these parameters are not cut I stone and that we have
pregnancies with 4 cell embryos on day 3. Additionally, we assess the amount of fragmentation and
the evenness of the blastomeres in the embryo to implant. The following embryo has the features
listed above .
 Figure 6

Here is an 8 cell embryo with >50 % fragmentation and about 7 8 cells. Sometimes with increased
fragmentation it is difficult to assess the blastomeres.

Figure 7

Here is an 8 cell embryo with about 20% fragmentation. You can see that the blastomeres are more
easily seen.

Figure 8

We also examine each of the blastomeres in the embryo to see if they are normal. If we see
multinucleation (more than one nucleus per blastomere) then we know that the embryo is
chromosomally abnormal. It is important to check at the appropriate time. Embryos that are
chromosomally abnormal may still develop into blastocyst stage embryos that have normal
morphology. Some examples are below.

This two cell embryo has multiple nuclei in each of the blastomeres. You can see them in each cell.
 Figure 9

When we followed this embryo in culture out to the blastocyst stage (next photo), it produced an
embryo that appeared normal, yet clearly was not. This is why it is important to examine they embryos
closely.

Figure 10

As we mentioned above, there are many beautiful and healthy babies born from embryos that were
not perfectly formed. We feel that if we are able to transfer embryos that patients ultimately have a
chance to conceive. The following cases support this. Also, if we do a procedure and the patient fails
to get pregnant, we will very likely change the protocol;

Case 1.

On her 1st IVF cycle she had a single 12 cell embryo and did not get pregnant. We changed the
protocol and on her second cycle she had two 4 cell embryos that were grade 1. We did assisted
hatching to help with implantation. She conceived twins and one split giving her three daughters
which were delivered and are healthy. Thus, we do have pregnancies with 4 cell embryos.

Figure 11
Case 2.

This patient did an IVF cycle using the long lupron protocol and produced three embryos on day#3
that ranged from 2-4 cells. She did not get pregnant. The embryos are shown below:

Photo 12

We changed the stimulation protocol and she produced much improved 7-8 cell embryos and
delivered a healthy child. The embryo pictures are shown below and you can see the improvement.
Thus, even though a patient may not do well initially, we often can change the protocol and have a
good outcome. We critically analyze each cycle to learn how we can improve.

Photo 13

Case 3.

This patient had approximately 50% fragmentation in an 8 cell embryo and delivered a very healthy
child.

Photo 14