HUMAN IN VITRO FERTILIZATION (IVF) TECHNOLOGY
D. Sakkas and *A.O. Trounson
In vitro fertilization techniques were initially adopted for the treatment of couples in which the female suffered from inoperable tubal blockage. Following the initial birth of Louise Brown in July 1978 (24), and the subsequent births that followed, it became evident that a wider range of infertility problems could be treated by in vitro fertilization (IVF). Today, the primary indications for treatment by IVF are absence of a functional fallopian tube, poor sperm quality and refractory anovulation. In all these indications the current technology available has increased the options and chances of success.
New applications in the treatment of infertility
Patient treatment has improved as a result of both scientific and clinical research. Improvements have been made in all the stages of treatment, commencing from the initial stage of induction of ovulation to the final step of transfer. For the induction of ovulation the use of GnRH analogues is now widely in practice to synchronise follicular development avoiding a precocious LH surge from leading follicles (15). As a possible alternative to the need for induction of superovulation the possibility now exists to recover immature oocytes from unstimulated ovaries. Cha et al. (3), successfully matured immature oocytes in vitro, fertilized them and then transferred the cleaved embryos leading to a viable pregnancy. Furthermore, in the field of hormonal therapy, there is now a treatment option available to agonadal women, or women suffering from ovarian dysgenesis or premature menopause. In these women, who receive donated oocytes or embryos, a sequential hormone replacement therapy can mimic the normal menstrual cycle followed by further hormonal support during the first 8 weeks of pregnancy (13,21).
IVF also permits a new access to gametes and embryonic material which broadens its application to domains out of its initial field of application. A number of studies have been performed on chromosomal abnormalities in oocytes, while embryo culture studies have been able to improve the development of embryos to the blastocyst stage in vitro (17, Sakkas, unpublished data). It has also been demonstrated that the embryonic genome of human embryos is activated during the 4- to 8-cell stage (1).
While the range of applications of in vitro fertilization now cover a wide spectrum, this chapter will predominantly deal with two techniques, one that is utilized to treat severe male infertility and the other used to diagnose for genetic defects in the preimplantation embryo.
Male factor infertility
The techniques utilized in the treatment of male factor infertility are dependent on the severity of semen parameters. Treatments can range from the use of surgical procedures such as gamete intrafallopian transfer (GIFT) to the use of micromanipulators that enable the direct placement of a spermatozoa into the perivitelline space or cytoplasm of an oocyte.
The World Health Organization guidelines state that the minimum semen parameters for defining a normospermic male are greater than 20 million sperm per ml, a motility of greater than 40% and greater than 40% normal morphology of sperm (26). In respect to IVF the choice of treatment that will be applied to a couple in which the male has reduced semen parameters depends largely on the amount of motile sperm that can be harvested. The choice of treatment can also depend on if the couple has had previous failures to achieve fertilization in an IVF treatment cycle and other factors such as failure of sperm to bind to the zona pellucida of the oocytes.
The technique adopted for the treatment of a male factor patient should attempt to utilize the least drastic measure of invasion on the oocyte. If the female has functional fallopian tubes then the GIFT procedure can be performed, while if enough sperm can be harvested, the oocytes should first be inseminated in a reduced volume. Failure to achieve fertilization should therefore lead to one of the micromanipulation techniques, again depending on the number and normality of sperm available. The three techniques in current use are partial zona dissection, sub-zonal sperm insertion and direct cytoplasmic insertion.
Gamete intrafallopian transfer (GIFT)
In the utilization of GIFT, the sperm are directly placed in the fallopian tube along with the oocytes, subsequently increasing the chance of sperm attaching to and fertilizing the oocytes. In a randomised trial of 100 male factor patients, that were allocated to either GIFT or tubal embryo stage transfer (TEST), Calderon et al. (2) found that the pregnancy rate per transfer was 25% for GIFT patients and 7% for TEST patients. In these cases the benefit of GIFT may be two fold. Firstly, placement of both the oocytes and sperm may lead to an actual increase in fertilization as specific tubal factors may improve the performance of the sperm as compared to in vitro. Experiments conducted on the effect of oviductal cells on sperm behaviour in a number of species support this argument (14,19). In one such study, Holden et al. (11) have shown that a factor of greater than 100kd secreted by human oviductal cells in vitro enhanced the motility of human sperm. Secondly, the preimplantation development of the embryo may be improved so that it is more viable. This second point is supported by the data of Yovich et al. (27) who reported higher pregnancy rates in a series of patients who displayed male factor characteristics and antispermatozoal antibodies that had pronuclear stage embryo transfer (PROST) compared to a similar group of patients treated by IVF.
The use of micromanipulation to assist fertilization
The main micromanipulation techniques currently in use are partial zona dissection (PZD) which creates a slit like incision in the zona pellucida and is followed by incubation of the manipulated oocyte in an insemination droplet (Fig. 1), sub-zonal sperm insertion which requires the insertion of one or more spermatozoa into the perivitelline space (SUZI) (Fig. 2), and, the microinjection of sperm directly into the cytoplasm of the oocytes termed intracytoplasmatic sperm injection (ICSI) (Fig. 2).
SUZI and PZD have been adopted by several clinics and have resulted in numerous pregnancies (5,6,16,18,23) (Table 1). The injection of sperm directly into the cytoplasm, ICSI, has been more limited in its application but has also resulted in clinical pregnancies (20) (Table 1).
The suitability of the micromanipulation techniques is related to the type of male factor patients under treatment. As previously mentioned, patients that fall into the category of male factor according to WHO guidelines can differ in their severity and therefore in the type of treatment applicable. The limiting criteria in treating male factor patients is the amount of sperm available after preparation. Initial studies by Cohen and colleagues showed that PZD was a viable alternative when compared to IVF (4,16). However, the true benefit of PZD in comparison to insemination in microvolumes or to SUZI seems limited. In a study comparing the effectiveness of the PZD and SUZI techniques we found (22) that for male factor patients with an initial sperm count of more than 10 million sperm / ml no difference was observed in the fertilization rates when using either technique. Male factor patients with an initial sperm count of less than 10 million sperm / ml had a significantly lower fertilization rate when treated with PZD. In addition, PZD results in a higher incidence of polyspermic fertilization (5,16,22) as the number of sperm entering the perivitelline space can not be controlled as in SUZI.
In contrast to PZD, the SUZI technique allows the injection of sperm directly into the perivitelline space. This subsequently allows the treatment of more severe cases of male factor patients and allows a greater control against polyspermy. In our initial studies (23) we found that the optimal number of sperm injected under the perivitelline space when performing SUZI that increased fertilization, while not jeopardising the level of polyspermy was 4 sperm. Injection of between 5-10 sperm led to a 50% occurrence of polyspermy. Combining SUZI with immediate transfer of the microinjected oocytes into the fallopian tube in a procedure termed MIFT (microinjection and intrafallopian transfer) has been shown by the Monash IVF research group in Melbourne to more than double the pregnancy success rate of SUZI (Calderon et al. unpublished; McLachlan, unpublished). This makes SUZI a particularly successful treatment and further suggests there is a benefit provided by the fallopian tubal environment, on promoting fertilization. One candidate for this increase in fertilization may be plasminogen which has recently been shown to improve in vitro fertilization rates in the mouse and is abundant in the oviductal fluid (12). In the MIFT technique 1 to 4 microinjected oocytes are transferred to the fallopian tube using a GIFT catheter and the average pregnancy rate per patient treated is around 25% compared to 11% per patient treated for SUZI and transfer of embryos to the fallopian tube (TEST) or uterus.
The injection of sperm directly into the cytoplasm of the oocyte allows the treatment of the most severe cases of male factor infertility. Unfortunately, this treatment is also associated with a higher risk of damage to the oocyte. Current results indicate that between 10 and 25% of the oocytes are damaged during ICSI (Van Steirteghem, personal communication; Sakkas, unpublished results). Therefore there is a greater impact on patients that have a small number of oocytes recovered at oocyte pick up. However, high fertilization rates (50-65% of oocytes injected) and low rates of oocyte lysis are possible and this has been achieved by Van Steirteghem and colleagues in their clinical IVF program (unpublished data). The pregnancy results of a number of European groups performing the three micromanipulation techniques are shown in Table 1.
Preimplantation genetic diagnosis
One application of IVF, out of the initial domain as a treatment of infertility, is the area of preimplantation embryo diagnosis. This technique aims to avoid the patient’s ordeal about the decision to undergo an abortion once an affected fetus is diagnosed by the more conventional techniques of prenatal diagnosis, such as amniocentesis or chorionic villus sampling. The technique takes advantage of the access to embryonic material with the aim of diagnosing a chromosomal or monogenic aberration in an embryo. This, theoretically, allows the transfer of non-affected embryos back to the patient.
The technique involves the removal of 1 or 2 blastomeres from an 8-cell stage preimplantation embryo for genetic analysis. The removal of up to 2 blastomeres has been shown to be of no detriment to the preimplantation development of the biopsied embryo (10). The following analysis of the biopsied blastomeres subsequently depends on whether the couples are at risk for a sex linked or genetic disease. The biopsied blastomeres can then be sex typed by fluorescent in situ hybridization, X/Y sequence amplification for a sex linked disease or genotyped by the polymerase chain reaction for an embryo at risk for a monogenic disease.
The technique of preimplantation embryo diagnosis is being clinically applied. Handyside et al. (8) reported the selective transfer of female embryos in five couples at risk of sex linked diseases that resulted in two twin and one singleton pregnancy. A number of births have also followed including the birth of a normal girl following testing for cystic fibrosis (9). Although a number of births and successful analyses have been reported, the rapid application of this technique is cautioned as it is still subject to misdiagnosis, even in experienced hands. The misdiagnosis of embryonic material has already been reported in two separate cases, one involving a couple at risk of an X-linked disease (7) and in one diagnosing the presence of the deletion of the gene causing cystic fibrosis (25).
Problems associated with the advancement of in vitro fertilization technology
IVF technology has broadened so as to offer a greater number of treatment options for patients, firstly, in the area of infertility and secondly, in areas such as the identification of genetic disease. Techniques such as oocyte donation allow women the possibility of increasing their reproductive lifetime past that of menopause, while in vitro maturation of oocytes could lead to an increase in the number of embryos a patient can produce without risking hyperstimulation. In addition, the manipulation of oocytes may possibly allow the sexing of embryos for other than genetic analysis and the eventual possibility of gene therapy. The advancements in human IVF technology increases the options for treatment of infertile patients and those with inherited severe genetic disease. However, these developments will also test moral and ethical attitudes within the community.
Edited by Aldo Campana,