Science Pulse
Abnormal Fertilization
Twenty-four hours after undergoing an egg retrieval procedure, a patient will receive a fertilization report. Occasionally the report will indicate that some of their eggs fertilized abnormally. This article explains the process of scoring fertilization and explains the results.
During an egg retrieval procedure, an embryologist, with the aid of a microscope, quickly identifies and takes custody of the eggs. They are immediately placed inside an incubator in our laboratory where they mature for 4 hours before being introduced to sperm. Then, depending on the quality of the sperm, an embryologist will either incubate each egg in the presence of 100,000 sperm, or inject a single sperm into each egg (in a process called Intracytoplasmic Sperm Injection or ICSI).
The next morning, 22 hours after the egg retrieval, an embryologist observes the eggs and records their fertilization status. A small percentage of eggs will be immature or non-viable. The remainder can fertilize normally, fail to fertilize or fertilize abnormally.
As fertilization gets underway and the sperm is working its way into the egg, the egg is busy completing the process of duplicating, then ejecting half of the female DNA. This reduction in DNA content, a process called meiosis, is completed only when the egg is fertilized; the sperm actually stimulates the egg to finish meiosis.
Meiosis happens in two distinct stages in eggs. The first stage is completed in the hours before ovulation or before a retrieval procedure, and readies the egg to receive the sperm. The egg discards some DNA in a small structure called a polar body (PB), and the presence of this 1st PB tells us that the egg is mature and can be fertilized (see Figure 1). The second stage of meiosis is very similar to the first, and it generates a second PB, but an egg will only complete this stage if penetrated by a sperm (both polar bodies are visible in Figure 2).
Once inside the egg, the sperm head swells and forms a structure called a pronucleus. The term “pronucleus” is used because it only contains half the DNA of a normal human nucleus. The egg responds by completing meiosis (ejecting the 2nd PB) and creating another pronucleus. The egg now has the right amount of DNA needed to create a new individual (˝ from the male (sperm) and ˝ from the female (egg)) and the 2 pronuclei are clearly visible in the center of the egg (see Figure 2). The egg has fertilized normally and this is confirmed by the presence of 2 polar bodies.
If we don't see any pronuclei (Figure 1), we assume that fertilization has failed. In this instance the sperm doesn't show up as a pronucleus inside the egg, and in the absence of a sperm, the egg doesn't complete meiosis. This lack of pronuclei indicates fertilization failure, and the presence of just a single PB confirms this finding (Figure 1).
While performing fertilization checks, the embryologist will observe either no pronuclei or 2 pronuclei in the vast majority of eggs. However, a small number of eggs, usually around 5% of all eggs inseminated, will show some number other than none or two pronuclei (see Figures 3, 4 and 5). These are the eggs that are abnormally fertilized.
Having more than 2 pronuclei usually suggests that more than 1 sperm managed to enter the egg. If this happens, there is one nucleus from the egg and one nucleus for each sperm that got in. An egg penetrated by 3 sperm for example, will have 4 pronuclei (see Figure 5). Please note that the egg has 3 separate mechanisms to prevent penetration by more than one sperm, so eggs with >2 pronuclei are uncommon.
Occasionally, an egg into which we only injected one sperm will show up with 3 pronuclei (Figure 3). When this happens we suspect that the egg did not complete meiosis and in fact retained the ˝ of the female DNA that it was supposed to discard in the 2nd PB. Such an egg would have 2 maternal pronuclei, and one paternal pronucleus. Regardless of the source of the extra nucleus, any number of pronuclei above 2 is abnormal and the resulting embryo is anomalous by virtue of having too much DNA, and it cannot result in a baby.
The other abnormality that we see is the presence of only a single pronucleus in the egg (Figure 4). This particular situation can arise in a number of ways, but it most often arises because the egg attempts to develop into an embryo without fertilization by a sperm. This process is called parthenogenesis, and without any male DNA, the embryo will typically die within a few days. Alternatively, if there's a slight acceleration or lag in the appearance of a pronucleus, we might only see one nucleus when we look. Genetic studies have helped in understanding the status of single pronucleate embryos: If the condition arises after sperm injection, where we know the precise time of sperm entry, up to 90% of the resulting embryos are abnormal. However, if a single pronucleus is seen after a normal insemination, where we don't know the exact time of sperm entry, or the maturity of the egg at insemination, over 60% of these embryos turn out to be normal.
We find that viable embryos mainly result from eggs with 2 pronuclei. Embryos arising from eggs with more than 2 pronuclei, or from eggs that were injected and had only a single pronucleus, are discarded as they are almost certainly genetically abnormal.
But fertilization is a transient process between egg and embryo. Pronuclei are only visible in the egg for a few hours and it is possible to miss one or more of them. Hence, we occasionally have embryos resulting from eggs where we didn't observe any pronuclei, or where we saw only a single pronucleus after incubating the egg with sperm. In these embryos, we can't be certain that they are normal. Typically, we keep them in our laboratory for at least an additional 2 days and watch their development carefully. If they behave like normal embryos, we can transfer them or freeze them for later use. It is important to note that we would only use these embryos if a patient had few or no normally fertilized embryos. If after transfer these embryos turn out to be abnormal, they would not implant and thus not result in a pregnancy.
When examining eggs for pronuclei, the embryologist looks very carefully at the pronuclear and polar body status of each individual egg, using an inverted microscope that can magnify up to 400 times. Small clues, such as the subtlety large size of the pronucleus in Figure 4, can explain what is happening. This egg is abnormal because the nucleus is too big to have come from a sperm. Without a sperm to fertilize the egg, it cannot complete meiosis and expel the 2nd polar body. The nucleus is therefore bigger than normal as it contains the DNA that an egg normally retains, plus the DNA that should have been discarded in the 2nd PB. We would have no choice but to discard this egg.
We generally transfer embryos that show the normal 2 pronuclei when observed on the day after egg retrieval. We are careful only to discard embryos that we see for certain are abnormal at fertilization. If for any reason we are unsure, we will give an embryo the benefit of the doubt if it displays normal growth patterns after fertilization.
We give patients their individual fertilization information on the day after their retrieval and discuss it again on transfer day. The hope is that you will clearly understand the fate of each of your retrieved eggs and we allow ample time for discussion if you have questions.
- Joe Conaghan, PhD, HCLD
Joe Conaghan, PhD, HCLD, PFC's ART Laboratory Director, is internationally recognized for his work with embryos. His background includes involvement in the first PGD on human embryos. His high standards and extensive experience brings national recognition to our laboratory. He also trains fellow embryologists for licensure and is an inspector for CAP, the licensing body for IVF laboratories in the USA.
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