Since the birth of the first baby achieved through conception outside of the human body in 1978, the principles of "in vitro" (literally "in glass") fertilization and culture have remained the same - careful establishment and maintenance of a well-controlled, sterile environment in which the normal physiology of fertilization and early development can be played out relatively undisturbed to provide healthy embryos for transfer back into the body. During the ensuing two decades, much has been learned, however, about the tolerances of such a system and how this technique can be exploited to treat a widening range of infertility cases. There have been great strides made in development of more appropriate culture media that has enabled embryos to be grown for extended periods of time in culture. Surplus embryos and possibly eggs may now routinely be cryopreserved in liquid nitrogen for use in subsequent attempts at pregnancy. Fertilization itself is no longer a hit-and-miss affair with the advent of assisted fertilization through micromanipulation. Embryos can be micro-manipulated for cell biopsy to determine their genetic status as well as aid in their ability to implant through drilling into their outer shell (assisted hatching).
CONVENTIONAL IN VITRO FERTILIZATION (IVF)
Through the controlled application of ovarian hyperstimulation, it is current practice to time the retrieval of mature oocytes (eggs) from a woman's ovary. The yield may vary anywhere from one to 30 or more eggs that may be retrieved depending on the responsiveness of the ovaries to the gonadotropins used to stimulate them. These eggs are gathered by the embryologist into an appropriately balanced salt solution and maintained at body temperature (37°C) until such time as they are ready to be inseminated. Meanwhile, a sample of semen containing the sperm destined to be used for each specific set of eggs is collected and processed by cell separation techniques to provide as clean and active a sample of sperm (atozoa) as possible. A major emphasis of the IVF laboratory is directed toward guaranteeing that the correct sperm go with the right eggs through good labeling and check systems. Ultimately, following several hours in culture, eggs and sperm can be mixed and allowed to bind and fertilize in a relatively natural fashion. Depending on the quality and maturity of both eggs and sperm, it is common for fertilization rates to vary considerably relative to the original number of eggs collected. Twenty eggs retrieved in no way guarantees 20 embryos. Likewise, 20 fertilized eggs in no way guarantees that there will be 20 embryos of sufficient quality for both cryopreservation and fresh transfer to the woman's body.
Central to the question of how many embryos are actually utilized in any IVF treatment cycle is the period during which the embryos are cultured in vitro. This can be as little as one day, or up to seven in the case of blastocyst growth and transfer. Assuming that culture conditions are relatively optimal, there is less and less reason not to culture embryos throughout their pre-implantation stages to allow the embryos to "select" themselves for transfer or cryopreservation. The blastocyst is the term given to the very last stage of an embryo prior to it implanting into the endometrial lining of the uterus. The poorer the rates of blastocyst growth are, the more restricted the choice of embryo is at this stage of development. In any event, growth of any embryos to the blastocyst stage improves the level of discrimination of embryo viability available to the embryologist, and is key to reducing the numbers of embryos used for uterine transfer. The more confidence a clinic has in the viability of the embryos it transfers, the less need there is for multiple transfers of three or more embryos. Thus with the transfer of three or less embryos, the risk of multiple pregnancies is significantly reduced, in turn minimizing risks of pregnancy loss or fetal abnormalities common in multi-fetal pregnancies.
MICROMANIPULATION IN IVF THERAPY
Micromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass microtools.
MALE FACTOR INFERTILITY
Micromanipulation first saw clinical use in IVF for purposes of assisted fertilization in the treatment of male factor infertility, where fertilization potential was low in cases of poor sperm quality. The ultimate evolution of this approach has been the development of the single sperm injection procedure referred to as Intracytoplasmic Sperm Injection, or ICSI. Sperm of virtually any quality and from any level of the male reproductive tract may be used with the only criterion for use being that the sperm is alive even if it is not moving (motile). Dead sperm may be able to achieve fertilization; however, the DNA or genetic material from such sperm is too degenerate to form a viable embryo. Immature sperm from the testicle or the epididymis can be retrieved for use with ICSI for men who possess no sperm in their ejaculated semen (azoospermia). This azoospermia is either due to an obstruction in the tract (obstructive), or to extremely low production of sperm in the testicle itself (non-obstructive). In certain cases, men may produce sufficient sperm, but they do not survive to the point of ejaculation (necrozoospermia). Consequently, instead of using non-viable sperm from the ejaculate, testicular biopsy will provide a ready source of freshly produced viable sperm.
INTRACYTOPLASMIC SPERM INJECTION (ICSI)
With the almost unlimited potential to achieve some level of fertilization with ICSI regardless of sperm quality, it would seem that male factor
infertility would no longer be of concern. It must be noted, however, that sub-fertility in men can be related to certain numerical and structural defects of the chromosomes and, therefore, there is a strong recommendation for all couples that achieve pregnancies from ICSI to undergo prenatal screening. In certain cases of obstructive azoospermia, there is a higher incidence of cystic fibrosis in the male. Hence, before embarking upon treatment of the more extreme forms of male factor infertility, it is advisable to have some cytogenetic screening performed. Incidentally, very subtle compromise in sperm quality may well be responsible for a marginally lower embryonic viability rate and a slightly higher early miscarriage rate even if such embryos implant. Such observations have led to the suggestion that the technique ICSI itself is at fault; but this misses the point that ICSI per se is not causing the problem, merely facilitating the use of sperm, which under other circumstances would never have even achieved fertilization.
ICSI FOR NON-MALE FACTOR INFERTILITY
The use of ICSI is now routinely applied to a range of clinical situations wherever there is a possibility that conventional in vitro fertilization may be suppressed or not occur. Such situations include the following: idiopathic or unexplained fertility; hyper-responsive ovarian stimulation cases where egg quality may be reduced; post-thaw sperm samples that survive poorly; post-thaw egg insemination; generation of embryos for pre-implantation genetic screening where embryos "clean" from any extraneous contaminating sperm is needed; or, indeed, any case where there is an extreme need to maximize normal fertilization, for example, when a woman has only a few eggs retrieved. It is possible to "rescue" cases following complete failed conventional fertilization with ICSI. The viability potential of these "late-fertilized" embryos is approximately half of timely fertilized embryos; nevertheless, they do generate successful live births. ICSI has become such a common feature of IVF therapy that it is fast becoming the insemination technique of choice.
It has been proposed that a certain number of otherwise viable embryos do not implant simply because they are unable to break free from the surrounding "jelly coat" (zona pellucida) when they reach the blastocyst stage of development. Around an unfertilized egg there exists a transparent glyco-protein coat that acts to protect the egg and regulate normal fertilization by any penetrating sperm. This jelly-like coat continues to protect the early preimplantation embryo until, as a blastocyst, the embryo fills itself up with fluid like a water-filled balloon, pumping itself larger and larger until it ruptures and "hatches" from the zona pellucida. The embryo is now ready to make contact in its naked form with the endometrium and implant. Inappropriate ovarian environment due to advanced maternal age or other factors that may compromise the follicular environment may in certain cases render the zona pellucida thicker or tougher. Such IVF cases may benefit from the application of a form of micromanipulation referred to as "assisted hatching" In this process, the embryo has a hole made in the surrounding zona pellucida prior to transfer to enable it to "hatch" free from the zona pellucida more easily when it expands as a blastocyst in the uterus. This technique has never been unconditionally proven to be effective in any well-defined group of IVF patients, and as such remains essentially an experimental procedure. Holes in the zona pellucida may be made mechanically, chemically, or by laser. With the advent of more routine transfer of blastocyst stage embryos, the future of this technique, usually carried out on day three of development, may seem in question. Indeed, at the blastocyst stage in vitro, it may be most appropriate to dissolve off the entire zona pellucida prior to transferring naked embryos into the uterus. This could be considered the ultimate form of assisted hatching without the need for micromanipulation. Currently, however, assisted hatching can be easily performed using a infrared laser to create a hole in the zona pellucida that allows the embryo an easy means of escape when it is time to try and implant into the uterine wall.
Briefly, it is of relevance in any discussion of micromanipulation techniques to mention the potential to biopsy both eggs and embryos. This approach is known as preimplantation genetic diagnosis (PGD) and enables the screening of both the unfertilized egg by removal of the first polar body, or the fertilized multi-cellular embryo by removal of one or more cells either at the 6-12 cell stage or from the trophectoderm of the blastocyst. This material can be probed for both genetic mutations or gross chromosomal errors. This technology remains in its infancy and can be of profound importance clinically, but at this time only for cases with very clear medically-defined needs. The biopsy procedure requires very exacting skills of the IVF laboratory, and the egg or embryo is not entirely free of risk during the procedure. Hence, couples whose offspring have a high chance of inheriting a genetic disorder may have their embryos screened. Women who are at risk of generating eggs with a high risk of chromosomal anomalies can benefit from having their eggs or embryos screened for chromosomal normality. While embryos can have their sex determined through this procedure, the GRS team considers it inappropriate to do so except in cases of sex chromosome-linked disorders.
It is a privilege for any scientist or clinician to have access to the earliest stages of human development through culturing gametes and embryos in vitro. And, as such, it requires a high degree of ethical responsibility to provide as safe and optimal an environment as possible for these microscopic changes. While much has been done to maximize IVF pregnancy rates over the last two decades, it nevertheless remains to improve individual embryo selection to the point where we can routinely transfer only one embryo at a time, while being able to successfully and consistently freeze all surplus embryos of sufficient quality for later use in attempting pregnancy.