Intra-Cytoplasmic Sperm Injection (ICSI)

Introduction

For those of us professionals who work in the field of reproductive medicine and assisted reproductive technology (ART) there can be no more significant milestone than the introduction of In Vitro Fertilization (IVF).

In 1978, in what are now regarded as rather primitive conditions, British doctors Patrick Steptoe and Bob Edwards achieved a medical feat that would not only make them famous, but also break scientific ground, challenge assumptions about human reproduction, and create a social and political stir that would carry over into the twenty-first century. These two medical mavericks cultured the first-ever human embryo and successfully transferred it into the uterus of a woman, Mrs. Lesley Brown. Due to tubal blockages she had been unable to conceive. Her daughter from this experiment, Louise Brown, is the world’s first IVF baby and now 26 years old. The pioneering efforts of Steptoe and Edwards did more than give Mrs. Brown a child, though: they unearthed the capacity of reproductive medicine. Indeed, the first instance of IVF stands as an emblem of hope and possibility, as the ultimate example of what reproductive specialists seek to offer through their work today.

To say that IVF revolutionized the world of human reproduction would most certainly be an understatement; Steptoe and Edwards effectively catapulted reproductive medicine into the future. The technology that captured the attention of the whole world and excited the hearts of all women struggling with infertility continues to be heralded and refined by further great minds and academic institutions throughout the world. Although IVF was initially reserved for women with tubal problems, technological improvements allowed for expansion and the use of IVF in connection with many other causes of infertility.
Over time, specialists gained a better understanding of the techniques of ovarian hyperstimulation for harvesting eggs, of the delicate process of egg retrieval, and of the particular laboratory conditions and medium required for successful embryo culture.

Throughout the 1980s and early 1990s IVF success rates on the whole continued to rise. There was, however, one exception: IVF did not seem to be able to help those couples where the sperm quality was poor. In these situations, despite providing the best possible environment for the sperm and eggs, fertilization was just not occurring. In order for fertilization to occur, at least 50 to 100,000 good quality motile sperm are required per egg. These sperm collectively dissolve the cumulus cells around the egg, thereby facilitating a single sperm to enter the ooplasm of the egg and achieve fertilization. Various efforts were made to try and enhance sperm’s ability to fertilize the egg, yet none of these were particularly successful. For example, the first process to address the problems of male factor infertility was called Partial Zona dissection (PZD). Through the use of PZD, the zona pellucida, or the “shell” surrounding a woman’s egg, was opened using either chemical dissolution or a sharp instrument to file through the shell. Unfortunately this did not guarantee that a sperm would fertilize the egg, and sometimes after PZD too many sperm would enter the egg causing genetic abnormalities resulting in an abnormal embryo.

Photograph of a mature egg with the “cumulus” cells all around it.

Unsatisfactory results such as this set the stage for another medical breakthrough. In 1992, Dr.Jean –Pierre Palermo at the Brussels Free University in Belgium described a revolutionary procedure – the most significant to build upon what Steptoe and Edwards had achieved 14 years earlier. The technique now commonly known as Intra-Cytoplasmic Sperm Injection (ICSI) began as a laboratory accident of sorts, in which sperm were micromanipulated directly into the egg. In the first 4 cases normal embryos developed and pregnancies ensued. Since its promising beginning, ICSI has become a routine treatment for male factor infertility around the world.

Remarkably, the number of sperm required for egg fertilization dropped from hundreds of thousands for standard IVF to one viable sperm for ICSI. This led to the development of more aggressive techniques to surgically retrieve sperm for egg fertilization from men who under normal conditions had no sperm in their ejaculates.

How is ICSI done ?

ICSI is part of the whole IVF (In Vitro Fertilization ) process. During a normal menstrual cycle, only 1 egg is usually recruited to mature in a capsule of fluid called a follicle. The maturation time of the egg inside the follicle, is usually 10-14 days. Most follicles measure about 16 – 20 mm when the egg is mature. At this time during natural ovulation, the follicle ruptures and the egg is released.

During IVF, fertility drugs are used to stimulate the ovaries and recruit a bunch of eggs. So instead of just one follicle maturing, a whole number of follicles are stimulated to grow. These follicles are monitored by ultrasound, and when they reach maturity the eggs are surgically retrieved. For retrieval, a fine needle is passed through the vagina and into the ovaries, where eggs are aspirated from the follicles.

The eggs are cultured for about 3 to 5 hours for final maturation in the laboratory. Concurrently the sperm is prepared. Sperm may be collected from an ejaculated specimen of semen, or prepared from a previously frozen surgically retrieved specimen.

The eggs are then stripped of the cumulus cells (which are the sunburst array of cells around the egg) The cells are removed to assess the maturity of the egg, to better visualize the egg during the ICSI process, and to prevent the inadvertent injection of DNA from the cumulus cells into the egg. The stripping is done with an enzyme called hyaluronidase, which is normally found in the sperm heads. In normal fertilization it is this enzyme that allows the sperm to digest their way through the cumulus cells to the egg.

A tiny collection of sperm is placed in a viscous solution called “sperm catch” – and this slows down the motility of the sperm making them more easily assessed and caught. A healthy sperm is identified and then immobilized by striking the tail with a tiny glass needle called the injection pipette. The sperm is then aspirated tail-first into the same glass pipette. Using a high-powered microscope with sophisticated star wars type manipulation equipment, the egg is located and held in position by a holding pipette.

ICSI workbench The microscope is in the middle. The handles on each side of the microscope allow the embryologist to manipulate the egg and sperm on the microscope platform, and thereby perform ICSI.

The injection pipette is inserted into the egg thereby tenting the membrane – and gentle suction applied until the elastic membrane is broken. Once the membrane is broken the sperm can be deposited into the egg and then the injection pipette is removed.

Once the pipette is removed the egg will resume its normal shape and is then put into a specialized culture medium in an incubator. It is then assessed for fertilization 18 hours after the injection.

Diagram showing the egg being held in position to allow the injection pipette (needle) to deposit a sperm in the cytoplasm of the egg.

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This photograph shows a mature egg being held in position, and a sperm being injected directly in to it (on the right) using a fine glass pipette.

Who Needs ICSI ?

Indications for ICSI may include the following...

1. Where a previous IVF cycle has resulted in failed fertilization
2. If the number of sperm in the ejaculate is very low.(Oligospermia)
3. If the motility of the sperm is poor. (Asthenospermia)
4. If the morphology (shape) of the sperm is poor. (Teratospermia)
5. If there are no sperm in the ejaculate (Azoospermia), and sperm therefore have to be surgically retrieved from the epidydimis (MESA – microsurgical epididymal sperm aspiration) or testis (TESA – Testicular sperm aspiration)
6. If there are antisperm antibodies present.
7. If there are frozen sperm only available, with limited counts and motility. Sperm may be banked and frozen in cases where the sperm counts are very low, in men who need the sperm surgically retrieved (TESA, MESA) or in men who have banked sperm before chemotherapy for cancer.

Are there risks associated with ICSI ?

1. Egg damage

The first potential risk related to ICSI is the possibility that the egg may be damaged during the injection process. The way in which the cumulus cells are stripped from around the egg can potentially damage the egg, and here the experience of the embryologist becomes so important. It is also possible that if the cumulus cells around the egg are not completely stripped free, some of this material (i.e. DNA from the cumulus cells) could be injected into the egg along with the sperm. Great care needs to be taken to ensure that this does not happen.

2. Genetic risks

Some otherwise healthy men may carry a genetic cause for their low sperm counts. Such men would not normally be able to father a child. It is therefore possible that boys born after ICSI may inherit a problematic gene on the Y chromosome from their fathers, and thus also later experience fertility problems due to abnormal sperm counts. Some of these better-known abnormalities are referred to as Y chromosome microdeletions, and can be screened for before ART (Assisted Reproductive Technologies). Most of these microdeletions are otherwise harmless.

It does, however, appear that there may be a slightly increased risk of chromosomal abnormalities associated with ICSI. This specifically seems to be related to the sex chromosomes. The two possibilities for this include the chance that the sperm selected and used for fertilization is abnormal, or that the procedure itself may lead to an increased risk of sex chromosome disorders. Problems that may be related to sex chromosome disorders include increased risk of miscarriage with affected embryos, congenital heart disorders, behaviour and learning disorders, and adult infertility. The whole variety of “sex chromosome” abnormalities can be detected by a prenatal test called an amniocentesis. Therefore, all women who have ICSI should be offered an amniocentesis during their pregnancy. This risk would appear to be less than 1 %.

3. Congenital Abnormalities

There has been a suggestion that children born by ICSI may have a slightly higher rate of congenital abnormalities. Any child born – either by natural conception or ART - has a risk of a congenital abnormality. About 3 to 5 % of all naturally conceived newborns have a recognizable birth defect. There are a wide variety of common birth defects – some minor and insignificant, and some major and more serious. Common birth defects include such things as club foot, hernias, cleft lip or palate, skin tags, heart defects, cysts and so on. About 75 % of major congenital abnormalities can be detected by a prenatal ultrasound done at 18 weeks of pregnancy.

The evidence so far is reassuring in that ICSI does not appear to significantly increase the risk for congenital abnormalities. A U.S. report in 2000 showed the fetal malformation rate to be 1.7 % in 4949 ICSI pregnancies. In a Swedish registry an anomaly rate of 4 % was seen in 1139 ICSI pregnancies. Although a report from Australia in 2002 did suggest that there may be a higher rate of abnormalities in children conceived by IVF or IVF with ICSI, the numbers in this study are small. Studies are ongoing to try and address this issue.

Other factors that may increase the risk of Birth defects include the relatively advanced age of infertile couples, the underlying cause for their infertility, medications used during the IVF process, the factors associated with the procedures themselves, such as freezing and thawing embryos, the potential for polyspermic fertilization and the delayed fertilization of the oocyte.

4. Imprinting Disorders

Before addressing this issue it is important to briefly discuss genes and chromosomes. A gene is the part of the body that contains genetic material or DNA. Many genes together make up chromosomes.

Genes, either alone or in combination, determine what features (genetic traits) a person inherits from his or her parents, such as blood type, hair color, eye color, and other characteristics, including risks of developing certain diseases. Defects in genes or chromosomes may cause changes in certain body processes or functions.

We have already discussed the fact that some men with low sperm counts may have a genetic cause for the problem. These genes may be passed on to their children, who then may also in turn experience fertility problems.

At the moment of natural conception, the 30,000 genes in the DNA of the father must combine with the 30,000 genes of the mother. This results in 2 copies of every gene. Together these form the “master” program to build an embryo – cell by cell. Sometimes during this process the genes of the father are turned off allowing the mother’s genes to be in control, and at other times the mother’s genes are turned off allowing the father’s genes to rule.

Imprinting disorders arise when either the mother's or father's genes imprint themselves on the program in places where it was not their turn – the best analogy is that of two people, mother and father, talking at the same time, instead of taking turns. In other words, both copies of a gene are turned on when one of them should be silent, and the result is a genetic error that may cause a developmental disorder.

Therefore genomic imprinting is the gene regulatory mechanism whereby only ONE of two parental genetic traits are transferred to the baby.

Poor sperm quality, the environment in which embryos are cultured, and IVF/ICSI may all potentially affect imprinting. Some of the molecular defects associated with imprinting disorders include Beckwith – Wiedeman syndrome and Angelman syndrome.

It has been suggested that there may be a connection between IVF/ICSI and imprinting disorders, although this has not been substantiated.

5. Childhood Development

The last question to be addressed is whether or not ICSI may be related to any concerns about childhood development. This is very difficult to assess because so many other factors can influence childhood behaviour and development. A number of studies have been done to try an address this issue. Overall the studies are reassuring. Although one study suggested that there may be a difference between the development of naturally conceived and ICSI children, two others found no difference at all. A recent study in Europe studied the development of 1000 IVF and ICSI children, and compared them to children born naturally. At the age of 5 years all the children were at the same stage in terms of IQ, physical growth and emotional development.

In summary, like any medical form of intervention there are risks and benefits. IVF/ICSI does have risks, but for most couples it constitutes an acceptable and effective therapeutic modality.

ICSI – the Future?

ART programs collectively continue to monitor the children born from such techniques. As already mentioned, this technology appears to be safe, though with a small recognized potential risk. At the present time the fertilization rate from ICSI is in the region of 65 to 70 % with fresh sperm, and about 50-60 % with sperm that has been surgically retrieved, frozen and thawed. Currently the results are poor with sperm that do not move, as it is still difficult to determine whether they are dead or alive. Current specialized techniques, which are used to determine whether sperm are actually alive or dead, may actually damage the sperm in the process. Better methods are being developed to assess immotile sperm to allow selection of the healthiest possible sperm for ICSI. There is also further research being done using immature sperm that are retrieved from the testicular tissue of men who have no mature sperm at all. In this instance, research is exploring ways to extract the genetic material from the nucleus of the immature sperm, and then injecting this into the egg. This process is referred to as ROSNI (round spermatid nucleus injection)

Conclusion

ICSI is a revolutionary and now 12-year-old technique to help infertile couples undergoing in vitro fertilization due to male factor infertility. It involves the injection of a single selected sperm directly in to the cytoplasm of the mature egg using a glass needle.

It is an effective process, however there are some definite risks. Research is continuing, and the process is being better understood and refined every year. Medical science recognizes that interfering with nature, whether with reproductive technology, birth control, immunizations, treatment of cancer etc. may all have consequences. At this time the data suggest that ICSI is an acceptably safe procedure, helping hundreds of thousands of couples achieve a healthy family.