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Louise Brown was 21 in 1999. Since her birth, in vitro fertilisation (IVF) has become a widely used treatment for the subfertile couple. Currently about 1% of births in the United Kingdom follow conceptions in vitro. Certain forms of subfertility, largely those derived from male problems (affecting up to 40% of subfertile couples), cannot be treated by conventional IVF, and the development of intracytoplasmic sperm injection (ICSI) has allowed some of these couples to conceive.
What is ICSI?
ICSI was developed in humans in Belgium in 1992.1 The procedure involves injecting a single sperm into an egg using a micropipette one fourteenth the diameter of a human hair. The spermatozoa can be obtained either after ejaculation or after aspiration (directly) from the testis or epididymis (percutaneous epididymal sperm aspiration). The spermatozoa are prepared by washing away seminal plasma and, where possible, separating the progressive (most) motile sperm from cellular debris. Poorly motile or abnormally shaped sperm are not usually selected for injection, unless no normal appearing sperm are available in the preparation. Progressive motile sperm are slowed down in polyvinylpyrrolidine, which increases viscosity of the medium and permits a better spermatozoon selection. Immobilisation is performed by crushing the tail of the spermatozoon with the injection pipette. This disturbs the membrane potential, appears to improve fertilisation, and prevents the tail of the sperm damaging the ovum cytoskeleton. If apparently normal fertilisation occurs, up to three of the resulting embryos are transferred to the uterus 48 hours after egg collection using a standard procedure in which a fine flexible catheter containing the embryos is passed through the cervix into the uterine cavity, and the embryos are expelled in a minimal quantity of medium.
Use of ICSI
ICSI is a major adjunct to conventional IVF and has been rapidly introduced world wide. More than 100 centres in the United Kingdom and more than 750 centres in the European Union are now performing ICSI (figures from the Human Fertilisation and Embryology Authority/European Society of Human Reproduction and Embryology).
On the basis of 1997 birth rates and assuming that 25% of IVF procedures involve ICSI, at present, there are some 10 000 “ICSI births” a year in the European Union. The use of ICSI is increasing so much that, in Belgium, as many as 60% of IVF cycles involve an ICSI procedure. One of the main reasons for the popularity of ICSI is that couples who are paying for treatment believe that their “take home baby rate” will be higher if ICSI is performed (for male factor problems), although this has not been confirmed by a randomised controlled trial.2 Increasingly ICSI is used for “non-male factor” problems such as tubal malfunction or “unexplained infertility” where fertilisation was poor or failed with normal IVF. This is in addition to the standard indication of oligozoospermia (often with coincident asthenozoospermia (poorly motile sperm) and teratozoospermia (abnormal forms)). More recently other advances in reproductive technologies have resulted in still further potential applications for ICSI (discussed below).
Why are there concerns about the safety of ICSI?
There is no suitable animal model—that is, an infertile primate—on which to test the technique, so the safety of ICSI could not be assessed on animal models before introduction. ICSI involves bypassing sperm natural/competitive selection by the use of a single spermatozoon.
The following concerns have arisen.
- The risks of using sperm that potentially carry genetic abnormalities: it is thought that oligozoospermic males carry a higher rate of genetic defects.3
- The risks of using sperm with structural defects: although there is no absolute evidence that teratozoospermia (abnormal phenotype) represents an abnormal sperm genotype, these sperm would not normally be those that fertilise.
- The potential for chemical and mechanical damage: chemical damage could arise from agents injected into the egg within the medium, including sperm slowing agents—for example, polyvinylpyrrolidine—or there could be mechanical damage to the ovum from the injection process.
- The risk of introducing foreign material into the oocyte: some culture media may contain heavy metals known to be toxic to sperm.4 The description of mammalian transgenesis by ICSI5 has shown the most convincing evidence (so far) that inadvertent transfer of exogenous DNA into the ova by ICSI could occur. Perry and colleagues5co-injected unfertilised mouse oocytes with sperm heads and exogenous DNA encoding a green fluorescent protein, with 20% of offspring expressing the integrated transgene. The risk of infection by exogenous gene expression or integration into ICSI embryos has also been inferred by the work of Chan and colleagues6 using rhesus monkeys. They have shown that exogenous DNA bound to sperm before insemination could be transferred to rhesus ICSI embryos, but was excluded from IVF embryos because of the sperm-egg interactions before sperm penetration.
There are new as well as continuing concerns. For example, Dowsinget al 7 have suggested the greater possibility of the transmission of trinucleotide repeat sequences from ICSI treated fathers to future generations. Excessive amplification of these trinucleotide repeat sequences is associated with the increased risk of neurodegenerative disease.8
Equally disturbing are reports by Schatten and colleagues9in Oregon using ICSI in rhesus macaque monkeys. In a standard ICSI procedure, the injection pipette is polarised at 90° to the (visible) first polar body. This is to avoid damage to the (invisible) first meiotic spindle, to which it has been assumed there is a fixed relation. Schatten has dismissed this assumption. Using fluorescent markers, he has shown that the relation between the first meiotic spindle and the first polar body is not fixed. Thus the injecting micropipette may damage the first meiotic spindle (with unknown consequences). It is possible that injection into the region containing the spindle could result in chromosome damage or chromosome misalignment.
What is known about outcome?
Most early ICSI programmes started in 1994–1995, and the eldest children are now only 5–6 years old. However, there are several early outcome studies on ICSI offspring. The large series by Bonduelle and colleagues10 11 has provided some reassurance. However, most of Bonduelle's reports lack a control group. Her work has suggested an increase in sex chromosome abnormalities in ICSI offspring,11 but this needs to be confirmed in a larger sample. Other reports12 13 about perinatal outcome of ICSI conceived children have been reassuring and include the recent report by Loft et al,12 which involved all Danish born ICSI children. Interim findings of a United Kingdom based population study14 have suggested that ICSI conceived toddlers are healthy in relation to a normally conceived control group. Less reassuring was the report by Bowen and colleagues15 suggesting that a single centre Sydney born cohort of children were developmentally delayed at the age of 1 in relation to a normally conceived control group. This study had a number of limitations including lack of power, multiple observers, unstandardised testing systems, and failure to allow for confounders.16
Severe idiopathic oligozoospermia (about 60% of ICSI treated patients in the United Kingdom) is now recognised in 10% of cases to be associated with specific gene deletions on the Y chromosome. Such deletions occur in the AZFc (azoospermia factor) region of the Y chromosome17 and other related genes. ICSI conceived boys from these fathers will inherit these Y chromosome microdeletions and will need ICSI themselves to become fertile unless there are further advances (as will their male offspring).
A European collaborative group involving Belgium, Denmark, Greece, Sweden, and the United Kingdom is performing a developmental study examining child and family welfare at school entry.
The best way to deal with the issue of congenital abnormalities is through a birth registry of ICSI children. In the United Kingdom, a birth registry is planned, and agreement in principal has been obtained from 98% of United Kingdom ICSI centres to collaborate.18
More recent developments in new reproductive technology
ICSI appears to be useful for other recent developments in fertility treatment where there may be a shortage of gametes.
EXTENDED EMBRYO CULTURE
In standard IVF, embryo transfer normally takes place at 48 hours, but embryo implantation rates may improve if the in vitro culture period is extended to five days—that is, with transfer taking place at the blastocyst stage.19
IN VITRO MATURATION OF OOCYTES/TOWARDS SINGLE EMBRYO REPLACEMENT
In another development, immature oocytes20 are being harvested and matured in vitro and then fertilised. This in vitro maturation may produce eggs of more certain quality than by the present practice of hormonally stimulated polyovulation producing ova of uncertain maturity. Better oocyte quality results in better embryo quality.21 At present, after hormonally stimulated polyovulation, these variable quality/maturity oocytes are harvested. These are then fertilised and typically two apparently normal embryos are replaced. In vitro maturation may obviate the need to replace two such embryos with the replacement instead of one better quality embryo.
Alternatively there are increasing advances in embryo scoring,22 which will allow the selection of a single better quality embryo after a standard procedure. These advances in turn may solve a fundamental problem of current IVF treatment, namely the birth of twins, triplets, and other higher order births.
ADVANCES IN FREEZING
Cryopreservation of oocytes is a technique developed to preserve oocytes of patients undergoing cancer treatment or for oocyte donors. Cryopreserved oocytes require ICSI for fertilisation (after thawing) because the cryopreservation process brings about changes in the zona pellucida preventing sperm penetration.2 Only cryopreservation of mature oocytes has been successful. There has been a first report of successful cryopreservation of postpubertal ovarian tissue and reimplantation into the same patient23 after previous oophorectomies.
IMMATURE GERM CELLS
This has led to speculation about the possibility of cryopreservation and subsequent reimplantation of immature, prepubertal germ cell tissue after children have been treated for cancer and also the separate possibility of the in vitro maturation of primordial germ cells. These are of unknown risk.
A mouse named Eggbert has been born after the first successful maturation from a primordial germ cell. Eggbert died young and was obese, diabetic, and had developed intestinal lymphosarcoma.24 Critically these problems developed after full physical maturity was attained.24
Although evidence to date suggests that ICSI conceived children are healthy, it is unsafe to draw any conclusions about their long term wellbeing. Caution needs to be exercised when considering the implications for potential children whose parents have conceived with help from these new reproductive technologies. Although infertility sometimes has devastating effects on a person's sense of completeness and self worth, the health of the child should be paramount in further developments of these new techniques.
I would like to thank Laura Hewitson (USA), Dagmar Gutnecht (Holland), Sue Avery (Cambridge), and Ben Lloyd who have commented on earlier drafts of the manuscript.