Embryology Laboratory Services

 

Egg (Oocyte) Fertilization

Eggs are fertilized in a woman’s body within her Fallopian tubes.  This event occurs within the first 24 hours after the eggs have been released from the ovaries and can occur only if there are sperm present around the eggs at this critical time.   The penetration of the egg by the sperm AND the resulting formation of the nucleus of the sperm and the nucleus of the egg within the interior of the egg is known as fertilization.In the Embryo lab, the eggs and sperm are combined by one of two processes, by insemination or by ICSI.

 With insemination, 15,000 to 30,000 sperm are placed in a small drop with one or two eggs.  The sperm enters the egg by dissolving a path through the outer shell, or zona pellucida, with an enzyme that is located on the head of the sperm.  This entry also requires vigorous swimming by the sperm until it gets through the shell.  At that point, the sperm and egg cell membranes chemically bind and the sperm is pulled into the interior, or cytoplasm, of the egg.  When the sperm enters the interior of the egg, its tail breaks off.  The significance of this is that the cell membrane that surrounds the head and the tail is broken.  The breaking of this membrane allows water to enter the sperm head from the cytoplasm of the egg.  Then, both the egg and the sperm form a nucleus within the egg.  These two nuclei are called pronulei until they merge to form the nucleus of the new embryo.  They each contain half of the chromosomes that the embryo will have.  The formation of these two pronuclei is what the embryologist looks for when they perform a fertilization check on the morning after the retrieval.  

With ICSI, a single sperm is injected into each egg.  This technique eliminates the need for the sperm to dissolve a path through the shell of the egg and to bind to the egg membrane.  And these are capabilities that some sperm do not have.   However, the placement of a sperm into the interior of the egg by ICSI does not necessarily result in fertilization.  This is because the nucleus of the egg and the nucleus of the sperm must form within the interior of the egg as a result of the sperm entering the egg.  Just like other events in the process of fertilization, this formation does not always occur. And just as with eggs that have been inseminated, the appearance of these two pronuclei is what the embryologist looks for when they check the eggs for fertilization the morning after retrieval.

Once fertilization has been confirmed, the eggs are put into culture dishes with a new type of media that will support their growth during the initial stages of division and growth.  This culture media mimics the tissue fluid that is present in the Fallopian tubes in the woman’s reproductive tract.
The newly-fertilized eggs are known as zygotes.  

No Fertilization or Abnormal Fertilization 

At fertilization check, the embryologist usually sees that some of the eggs are not fertilized.  These eggs fail to fertilize for a variety of reasons such as:


The embryologist may see that some of the eggs are immature.  As eggs mature, they go through stages of chromosomal division.  These stages result in a reduction in their chromosome number by one half.  The extra chromosomes are contained in a small, round body located on the side of the egg.  This is called a polar body.  If an egg does not have a polar body at the time of the fertilization check it is still immature and not ready for a sperm to fertilize it.  Also, if it has not formed a polar body by this time (twenty four hours after the egg retrieval), it is defective and will not be capable of fertilization at a later time.

There are some eggs that have a polar body present but are still not fertilized.  They are mature, but have not shown the characteristic formation of their pronucleus or the formation of the sperm’s pronucleus.  This can happen if the egg is defective in its number of chromosomes and does not have a complete plan for building an embryo.  This results in the egg being incapable of further development.  The sperm can also be the cause of a mature egg not fertilizing.  The semen sample may have a low number of sperm that are capable of penetrating the egg’s membranes.  This is often associated with a high percentage of abnormally shaped sperm.  In severe cases, this may result in total failure of fertilization.  If this is suspected, intra-cytoplasmic sperm injection can be used to overcome this penetration problem.  If the problem with the sperm is chromosomal in nature, fertilization can fail because the sperm does not have a complete plan for building an embryo.  In this case, it will not matter if the sperm penetrates the egg, because it cannot form a pronucleus.  The egg will not fertilize and it will prevent any further development.  Unfortunately in these cases, intra-cytoplasmic sperm injection cannot overcome the problem. 

There are some eggs that are in the ovaries that have no chance of developing into an embryo because they are atretic.  Atresia is a form of abnormal development in which the cytoplasm, or cell material, of the egg is dark and non-functional.  Just as there are sperm that have abnormal morphology and are not functional, so too there are eggs that are abnormal from the moment of their development and have no potential for growth. 


Some eggs have perfectly healthy chromosomes and cytoplasm but may have a poorly formed shell, or zona pellucida.  These eggs often break during the aspiration process that is necessary to remove them from their follicle.  The result is an egg that loses its shell or one in which the cell material, or cytoplasm, also breaks.  Without the protective shell at this early stage, the egg cannot survive and grow normally.  If these eggs with weakened shells had not been aspirated, but had been allowed to ovulate and enter the Fallopian tube, the result would likely be the same.  They would not have survived the fertilization process in the body and would not have developed a normal embryo without the protection of an intact, strong shell.

At fertilization check, the embryologist often sees some eggs that have abnormal fertilization.  These eggs exhibit abnormal fertilization for several reasons including:


The embryologist may see eggs at the fertilization check that have three or more pronuclei instead of the normal two pronuclei.  This can occur for one of two reasons.  When an egg is placed with several thousand sperm during the process of insemination, a mature, healthy egg will allow a single sperm to bind to its cell membrane and enter its cytoplasm.  Immediately, a chemical process occurs within the egg that will block any other sperm from entering, even though it is surrounded by thousands of sperm.  This is known as the cortical granule reaction.  Some eggs are defective in this process and the chemical reaction is slow or incomplete.  In these cases, a second sperm may enter the egg’s cytoplasm and form a third pronucleus.  This third set of chromosomes will add to the plan for building an embryo and completely confuse and shut down the process of embryo growth.  These abnormally fertilized eggs may grow for a few divisions and then stop, or worse if transferred, may grow to the point of early implantation, implant on the uterine wall and then result in a miscarriage.  These eggs are referred to as polyspermic eggs.  Because these abnormally fertilized eggs have no potential for producing a viable pregnancy, they are immediately discarded. 

When intra-cytoplasmic sperm injection (ICSI) is performed, the possibility for multiple sperm entering an egg is eliminated.  However, there are eggs that have been injected with a single sperm and not exposed to any other sperm, but still have three pronuclei.  The process which results in this outcome involves the division of the female chromosomes.  When an egg matures, it releases one half of its chromosomes in a polar body.  As a sperm enters an egg, by insemination or by ICSI, its presence is signaled to the nucleus of the egg.  This causes the egg chromosomes to align and to form a duplicate set of chromosomes which align next to the first set of chromosomes.  The two sets of chromosomes then exchange sections in a process that results in each set of female chromosomes having a different composition than they had before the crossing over occurred.  The final step in the normal process is for one half of the chromosomes to be expelled into a second polar body outside of the cytoplasm of the egg.  Sometimes this does not occur and the extra set of chromosomes is retained within the egg.  The result is a third pronucleus that has the same effect as if the source of these chromosomes was from a second sperm.  Too many chromosomes will prevent the egg from developing normally.  These eggs are referred to as polygynic or polyploid.  Because they have no potential for producing a viable pregnancy, they are immediately discarded.  Since this source of too many chromosomes can also occur in eggs that have been inseminated, the source of the third pronucleus is not always known.  For this reason, these eggs with more than two pronuclei are referred to as polyploid, or having more than two sets of chromosomes, regardless of the source.

Finally, another abnormal condition that is seen at the fertilization check is an egg with only one pronucleus.  This usually occurs when a defective sperm enters an egg signaling the egg to form its pronucleus.  Meanwhile, the defective sperm is incapable of forming its pronucleus.  With only one half of the chromosomes functional, the plan for building an embryo is incomplete and the process shuts down. These eggs are known as 1pn and have no potential for life.  They are immediately discarded.