Infertility: Intra-cytoplasmic sperm injection (ICSI), micro-assisted fertilization (MAF) and sperm retrieval techniques

Intra-cytoplasmic sperm injection (ICSI), micro-assisted fertilization (MAF) and sperm retrieval techniques

Background

For people relatively new to the field of In Vitro Fertilization (IVF) the term ‘Micro-Assisted Fertilization may seem a little strange. This term is still used by some centers, but the technique it now refers to has the acronym ICSI, which stands for Intra Cytoplasmic Sperm Injection. The substance of the egg is called cytoplasm, and intracytoplasmic sperm injection, as the name implies, is the injection of a sperm directly into the cytoplasm of the egg. This procedure by-passes all the natural barriers that the sperm has to encounter. It has been used by animal embryologists since the late 1950s as a tool to understand the mechanisms of fertilization. In recent times it was developed to procure conception and pregnancy, which resulted in live offspring in domestic and laboratory animal species. However, scientists are reluctant to use the ICSI approach for the treatment of infertility cause because it is so invasive. Instead, a range of other procedures which involve by-passing the outer shelf of the egg, but not penetrating the egg itself, had been developed – these have been given acronyms such as ‘PZD’ and SUZI”, but ICSI has now replaced them all.

The first clinical success of ICSI, in 1992, lead to worldwide adoption of this procedure as the ultimate micro-assisted fertilization approach. In Britain, in 1993, there were only three Units licensed by the Human Fertilization and Embryology Authority (HFEA) to utilize this procedure. In 1997 there are 47 units in the UK licensed to offer ICSI as it is now recognized as a treatment of choice for at least 30% of all couples requiring IVF technology. The HFEA has also recognized the importance of this procedure in particular, and the high level of skill and expertise required to perform it successfully. As a result, the HFEA now require individual ICSI practitioners to be specifically licensed by the Authority in addition to the Unit itself. Therefore, individual ICSI practitioners will take their license and ICSI log book with them should they move to a new clinic. (Patients attending a centre for ICSI specifically, may wish to inquire of the experience of the ICSI practitioners, they may wish to obtain evidence of the clinic/practitioner’s particular experience – this data should be available).

Who requires ICSI

There are two main groups of patients that may require ICSI.

Patients who have a severe sperm problem, which prevents them even attempting conventional or a modified form of IVF.
Patients who have previously attempted IVF but have failed to achieve fertilization (generally on more than one occasion).

In group 1, patients can be further subdivided into ‘routine’, complicated and surgical.

Routine

In this classification we have patients with a very low sperm count, very poor motility or a high percentage (greater than 95%) of sperm with abnormal shape (morphology).

However, the majority of men have a combination of these problems often associating all three.

Complicated

Amongst this group of patients, there are a range of difficult conditions which include men who do not appear to ejaculate any sperm (Cryptozoospermia). In this situation, we are able to use modified methods of sperm preparation, where we can concentrate the seminal plasma into a volume the fraction of a teardrop. Often we find just a few sperm, literally just as many (or sometimes less) as there are eggs. However, we only need one sperm per egg for SI. Other conditions include men who have a range of different abnormalities in all their sperm; for example, a condition called Globozoospermi, where the top half of the sperm head (the acrosome) is missing; or a condition in which all the sperm are immotile. In this latter condition, many of the sperm may be living, but the tail is unable to function. We now have methods of selecting the living sperm from the truly dead sperm. By this procedure we can individually select the living sperms and use them for ICSI.

There is also a large group of men who, for various reasons, have problems obtain-ing an erection and ejaculating. This includes men with spinal cord injuries, Hodgkin’s disease, diabetes and numerous others. There are a number of ways to approach this problem and one of which is a procedure using electrostimulation. In this procedure a probe is inserted through the rectum which provides gentle stimulation to the nerves which bring about ejaculation. Often millions of sperms can be recovered by this procedure, but their quality is too poor for anything other than ICSI. However, by utilizing the ICSI procedure the technology is extremely successful.

Depending on the individual’s situation anaesthesia may or may not be required. Should it be difficult to obtain sperm by electrostimulation, a surgical procedure could be utilized.

Surgical

A number of conditions fall into this cat-egory and these include obstructive azoospermia – where sperm is manufactured in the testicles but there is an obstruction in the reproductive tract preventing the release of sperm – con-genital absence of the vas-deferens (the tube that carries the sperm from the testes to the penis) – in which some men were born without either vas-deferens – and failed vasectomy reversal. In these cases sperm can be recovered from the network of tubing called the epididymis.

The epididymis are attached to the testicle and act as the immediate reservoir for sperm before they are transferred into the vas and ejaculated. Sperm can be recovered from the epididymis either by inserting a needle across the skin, which does not require surgery, this procedure is called Percutaneous Epididymal Sperm Aspiration (PESA); or, but now less common, a procedure called MESA (Micro-surgical Epididymal Sperm Aspiration) can be used. There are a number of cases where it is impossible to recover sperm from the epididymis, and occasionally this arises even though doctors have attempted the PESA or MESA approach in the first instance. It is now possible to recover sperm directly from the testicle either by using a needle to aspirate the sperm (TESA – Testicular Sperm Aspiration), or by doing a biopsy of the tubes (Seminiferous Tubules) that act-ually manufacture the sperm (TESE- Testicular Sperm Extraction).

Therefore, providing sperm is being manufactured in the testes, it can be obtained either from the ejaculate, the tubes leading from the testes or from the testes themselves, depending on the individual condition. If a mature sperm is obtained by any of these procedures, using ICSI means there is a chance to have a baby, even in the so called “impossible” situations.

Some couples have been classified as “unexplained” infertility because all the tests available have not found a significant problem on the female side or with the sperm. However, a considerable number of these patients (somewhere in the region of 20%) – will have a fertilization problem. This can be overcome by using ICSI. There are also a range of rare conditions with the male, which produce unusual sub-microscopic defects in sperm preventing fertilization. It is possible that these also can be overcome using ICSI.

In some couples there may be a problem with the outer shell of the egg, which prevents sperms either attaching to or penetrating the outer shell. Sometimes it is difficult to evaluate this condition, and it is paramount to ascertain whether the failed fertilization is related to an egg or sperm problem.

Patients should possibly consider a crossover donor sperm insemination for diagnostic purposes only. The use of donor sperm with the unfertilized eggs would give us an indication as whether or not it was an egg or sperm problem. This provides maximum information and does not waste opportunities for gaining know-ledge. This is very important as doctors need to assess whether ICSI or egg donation should be the considered route in the future.

The Procedure

The woman is stimulated for follicle (the tiny sack in the ovary which carries the egg) production, as in conventional IVF. Egg recovery is identical to that for routine IVF, but sperm treatment differs according to individual patient circumstances and, even if obtained from the ejaculate, often requires a modification of the procedures used for IVF. This modification uses a high centrifugation method to concentrate the contents of the seminal plasma into literally a tiny fraction of a teardrop from which just a few sperm can be obtained and extracted. In certain circumstances we might use our micro-injection needles (approximately 12 times thinner than a single strand of human hair) to isolate single sperms from the seminal plasma. Whichever method is used, centrifugation or single sperm isolation, it is essential that the sperm is washed free of the seminal plasma.

The human egg aspirated from the follicle is surrounded by thousands of specialized cells – the cumulus cells (these perform a ‘nursing” role while the egg is in the follicle). These cumulus cells are removed by treatment with an enzyme (hyaluronidase) – a natural enzyme that is produced by the sperm during its passage through the cumulus cells whilst in the fallopian tube. In less than a couple of minutes the enzyme digests away the cells leaving the egg encased in a few layers in another type of specialized cells. These are mechanically removed by the embryologist using gentle suction into a very finely-pulled glass tube. The egg denuded of almost all its surrounding cells is then accessible for ICSI.

The ICSI procedure per se begins by first immobilizing the sperm. This is often performed by transferring the sperm into a viscous solution, which dramatically slows down its motility. In its sluggish state the single sperm has its tail permanently immobilized – this has been shown to be an extremely important part of the process.

Sperm is then aspirated into the tiny micro-needle and carefully maintained at its tip. The micro-injection needle is manipulated using a micro-manipulator which has extremely fine control capabilities. The egg itself is held onto another micro-tool by gentle suction to keep it firmly posit-ioned. The micro-needle containing the sperm is pushed gently up against the outer shell (zone pellucida) and carefully pushed through the shell, through the outer membrane of the egg and directly into the centre of the egg itself, ie. the egg’s cytoplasm.

Once the needle is inside the egg, a tiny amount of cytoplasm is aspirated into the micro-needle to mix with the sperm and ensure that the egg has been properly penetrated. Despite the tiny size of the egg (approximately 7 times smaller than the average full stop), the membrane is a very elastic structure and can be extensively stretched without actually being ruptured, (I often liken this to poking a finger into a balloon – you can touch the other side but not rupture the membrane!). Once the embryologist is certain that the egg has been penetrated, the sperm and cytoplasmic mixture is injected back into the egg. This procedure rarely causes residual damage to the egg, and has no lasting effects on further development. The whole procedure is performed under a high powered microscope.

In some cases, immotile but living sperm is being used. It is therefore important for the embryologists to be able to distinguish between dead and living immotile sperm. In this situation a solution is used which causes the tail of a living, but immotile, sperm to curl. The curled tail indicates that the sperm is actually living. It is these that the embryologists are able to select with their micro-needle. Once isolated, they can then be transferred to the viscous solution and treated in the same way for the ICSI procedure.

At the end of the injection procedure the micro-injection needle is carefully withdrawn and suction on the egg is released. The egg is washed through a few changes of normal culture medium, and left overnight in an incubator at 37ºC in conditions similar to routine IVF culture. The subsequent culture procedures, checking for fertilization, cleavage of the fertilized egg and transfer of any embryos to the womb occurs in the same routine manner as that for conventional IVF.

Results and outcome

As mentioned above, ICSI can be used

for all types of sperm problems – even those with the most extreme condition. Taking into account even the most difficult cases, recent data indicates that 98% of all patients should achieve fertilization and the transfer of at least one embryo with the ICSI technique, and 30% of patients having embryo transfer should achieve clinical pregnancy (fetal heart on ultra-sound scan). Analysis of the international data, indicates that, overall, couples have the same incidence of implantation in pregnancy as those undergoing conventional IVF (ie. about 25% per treatment cycle started).

There appears to be a slight reduction in the incidence of clinical pregnancy in cases where 1. sperm manufacture – as opposed to sperm release (a condition known as non-obstructive azoospermia) – is severely compromised, 2. where living but immotile sperms are used, and 3. there have only been two reports of a successful pregnancy with the condition called Globozoospermia. In certain areas other specific conditions, such as the cause of immotile sperm due to Kartagner Syn-drome, there is a reduction in the incid-ence of fertilisation and pregnancy. How-ever, in these extreme groups the numbers treated are obviously few and statistical information is currently unreliable.

In patients who have congenital absence of the vas-deferenes, and in a few other situations, it is necessary for couples to be assessed for Cystic Fibrosis carrier status. It has been shown that in certain parts of the world as many as 70% of men with congenital absence of the vas-deferenes are carriers of Cystic Fibrosis.

Safety

There has been some concern as to the safety of the ICSI procedure. This concern has revolved around the incidence of congenital abnormalities at birth and the findings of the chromosome studies of the fetuses assessed in utero. The major studies that have been reported on nearly two thousand pregnancies, indicate that the overall incidence of congenital abnormality at birth is not significantly higher than after IVF or the normal population. However, there has been some concern as to a slightly higher risk of abnormal sex chromosomes (the X and Y chromosomes) arising after ICSI. More information has to be obtained to truly assess the risk.

It is very important for patients to appreciate that, in most of the abnormal conditions, we are unable to explain the cause of the problem. Men should be assessed for their chromosome status, but it is not possible in all circumstances to study their genes (the coded information carried by the chromosomes which deter-mine every aspect of our make-up). It is highly probable that in a number of men their sperm problems are caused by under-lying genetic problems. We now know that there is a certain gene on the Y chromo-some which, if affected, causes very low sperm counts. It is therefore probable that sons resulting from ICSI will carry the same genetic problem as their fathers and, therefore, might themselves require ICSI (or some other technique) for them to conceive. Hence, some men should have blood taken to screen their chromosomes, assess their genetic condition and their status regarding cystic fibrosis. Many centers counsel their patients with regard to amniocentesis (testing cells around the baby during pregnancy) after ICSI, especially if ICSI has been used successfully in a particularly rare situation. This is a sensible approach and one that needs to discussed with all the appropriate professional parties.

Therefore, despite the staggering success with ICSI, we must not lose sight of the fact that this procedure is still new and as such our excitement must be tempered with caution. At the moment, more than 5,000 babies have been born worldwide. Although the low incidence of congenital abnormalities is encouraging, we must adhere to screening programmes, not become complacent but continue to gather all the data on outcome of treatment and make available ail information to patients. Currently we believe that as many as 1/4 of patients may carry a genetic condition which could be passed on to their children.

The future

Until recently, it was assumed that the sperm used to procure successful fertilis-ation and birth had to be a mature sperm-atozoon. The only men we could not treat, therefore, were those who did not manufacture any form of sperm or only immature sperm. Recently, scientists have achieved the world’s first publication of a pregnancy using a spermatid (the immature sperm) using ICSI technology’. This technology has been continued in our associated centers internationally to try to obtain more information on the viability of this procedure’. However, the use of immature sperm in the UK is not approved by the HFEA at present. Research is continuing, as are discussions with the Authority on the possible use of immature sperms in the future. Worldwide there have been approximately 20 babies born using immature sperm, but these are case reports and it is not known how efficient this procedure is. Too few patients have been treated to provide reliable data on the incidence of pregnancy. All appropriate screening of patients will also be essential for those seeking this technology in the future.

ICSI technology has revolutionized the treatment of male factor infertility. This is considerable, because we now appreciate that male factor infertility is probably the largest single cause of infertility amongst couples. Although this is exciting technology, we are also aware that it requires considerable skill and expertise to provide a successful treatment. In centers that offer this level of skill, more than 90% of couples treated will have the same chance of a healthy pregnancy and delivery as in conventional IVF. Patients should embark on an ICSI program with the same enthusiasm as those requiring conventional IVF, but detailed discussions should be undertaken with the professionals with regard to the couple’s individual circumstances and their risks and opportunities.