Editors: Norio Fujiki, M.D. & Darryl R.J. Macer, Ph.D.
Dept. of Obstetrics & Gynecology, Nagoya City University Medical School, JAPAN
Prenatal diagnosis is now concentrating on the early diagnosis of disease, and now even diagnosis before pregnancy is possible. Prenatal diagnosis of genetic diseases has been made possible by the remarkable progress achieved in genetic biochemistry, cytogenetics, and other related fields. In the 1970's amniocentesis was developed to take fetal samples, and using these technologies prenatal diagnosis rapidly developed. However, promotion of amniocentesis in clinics gave rise to new problems, namely because of safety issues for the mother and fetus, it took place after 16 weeks of fetal development. That means the mother has to wait for 16 weeks, which creates a great deal of anxiety. If abortion is also to be considered there will be more anxiety, and because this is the period when the mother feels the presence of the fetus there could be enormous mental trauma. In order to eliminate the negative aspects, chorionic villi sampling was developed. In this, a sample of fetal cells from the chorionic villi can be taken, and we can get the same information as we can from amniocentesis. This chorionic villi sampling became very popular in western societies in the 1980's.
These advances have contributed greatly to the prevention of the birth of children afflicted with several severe genetic diseases. At present, amniocentesis, chorionic villi sampling, and fetal blood sampling are all directed to the fetus. If the fetus proves to be afflicted, the parents then have the option of terminating the pregnancy. However, the decision to induce an abortion is likely to inflict mental trauma on the parents, particularly when previous pregnancies were also terminated.
The applications of molecular biology to clinical medicine in the 1980's drastically changed the approach to genetic diagnosis. There were new findings, one after another, with respect to the human genome, which were transformed into clinical genetics. The study of genes is synonymous with the study of genetics. One can now identify genes and establish their normalcy or abnormalcy even before the transcription of their products. DNA analysis has the potential of allowing for diagnosis of all monogenic disorders prior to initiation of pregnancy. In particular, DNA amplification by the polymerase chain reaction (PCR) is one of the recent outstanding developments in this area. With this methodology, DNA obtained from even a single cell can be amplified a million fold. This has led to dramatic changes in molecular diagnosis.
Currently, considerable advances have also been realized in the field of reproductive medicine. Since the introduction of "reproductive technology", in vitro fertilisation and embryo transfer (IVF-ET) in the 1980's, the same period as the above genetic techniques were developed, IVF-ET have become routine clinical procedures with important applications in the treatment of infertility.
If we look at the twenty years of prenatal diagnosis, if we look at the 1990's, the focus of our attention will be preimplantation diagnosis. Amniocentesis and chorionic villi sampling are playing an important role in the prevention of the birth of infants with serious diseases, but these diagnoses are focusing on the fetus. Even if the diagnoses take part during the early part of pregnancy we may need to abort the abnormal fetus, which causes anxiety to the woman. However, if the diagnosis is done before pregnancy, then we will not need to worry about abortion. This is the main reason why preimplantation diagnosis was developed.
Because of the relatively easy procurement of early embryos as well as the use of DNA amplification by PCR in IVF-ET programs, genetic information can be obtained at an extremely early developmental stage prior to pregnancy. This type of preimplantation testing represents an epoch-making approach to prenatal diagnosis because it eliminates the need for pregnancy termination.
There are basically three types of preimplantation diagnosis. One type is genotyping the human oocyte, the second is embryo biopsy during cleavage, and third is embryonic biopsy at the blastocyst stage.
The ovum is ovulated after the completion of the first meiosis and there is an oocyte in which the first polar body can be observed. With genotyping of the human oocyte, we use the first polar body for genome analysis. The polar body diagnosis was first devised, knowing that the polar body was sometimes damaged in in vitro fertilisation, but it did not give an adverse effect to the development of the fetus or subsequent development. Therefore we could see the polar body may not play an important role. The genetic information needed for embryonic development would not be damaged by removing the polar body. The polar body is removed by a suction pipette.
In the clinical practice, the women is treated with hormone to make them superovulate, and then we can obtain about 5-10 ova. Then we can isolate the DNA from the sample, and may amplify it with PCR. If an abnormal gene is detected, its counterpart in the primary oocyte should be normal, assuming that crossing over did not occur at the time of nuclear division. Verlinsky of the Illinois Mesonic Medical Center succeeded in detecting the cystic fibrosis gene in the polar body, but they have not reached the stage of giving birth to a normal infant. Sometimes there will be uncertainties about the diagnostic result. The second polar body testing and embryonic biopsy are required for clarification. A clinical application for sickle cell anemia, a-thalassemia, b-thalassemia and cystic fibrosis and Duchenne muscular dystrophy.
Another method for preimplantation diagnosis is embryonic biopsy during cleavage. After fertilisation, the embryo repeats cleavage to move through the oviduct in about 8 days, by which time it has grown into a blastocyst of 200-300 cells, and it is then implanted in the uterus. In the case of embryonic biopsy 4-8 cell embryos are used.
To obtain embryos at at early stage we have to perfuse the oviduct 3-4 days after ovulation. In a normal cycle we can only recover one, so the efficiency is quite low, so we perform superovulation to obtain a large number, and after IVF we can normally obtain more than two embryos which will develop. The embryo is immobilised using negative pressure from a sampling pipette and 1-2 blastomeres are obtained. In Hammersmith Hospital, London, Dr Handyside and others have already used this successfully. They diagnosed five carriers of sex-linked recessive diseases, and the normal female embryos were returned to the uterus and full term deliveries were observed. They had transferred 17 embryos to five women, and achieved five babies, with a rate of success of 29%. In this case the rate of live births is equivalent to an IVF-ET programme.
We are now at the preparative stage of embryo biopsy, and have performed some experiments. There are two methods for removing blastomeres, one is the extrusion method and aspiration method. We had a higher success rate with the aspiration method, compared to extrusion, as far as the number of viable embryos is concerned. With extrusion, 91.9% of the embryos burst, compared to only 27.9% of the embryos after aspiration. We also varied the number of blastomeres removed. There was 86.3% viability after removing one or two blastomeres, but when four blastomeres were taken there was lower viability. For safety we think the number should be limited to two.
The collected blastomere is used for genome analysis. We use human embryos which we judged to be degenerative or not proper for embryo transfer to women, based on growth analysis. We used the DYZ1 region for amplification and checked whether the Y band was present, to determine the sex. We also used a Y probe for fluorescent in situ hybridization (FISH) analysis, and applied both methods to the same embryos. Almost all of the results matched each other. Although we have to study this further, either one has good accuracy in detection.
The embryo biopsy at the blastocyst stage involves immobilising the blastocyst, then making a tear on the surface, carefully avoiding the embryonic pole. Then herniation is performed on the trophoectoderm, and part of the herniated ectoderm portion is cut out and used for sampling. We can call this an extremely early chorionic villi sampling. This procedure was performed on animal embryos. Verlinsky's group is the only group to study this on human embryos. They used 18 triploid blastocysts, presumably due to polyspermy, and in 12 cases karyotype analysis was done. In 14 cases hemoglobin detection through PCR was possible. The inner embryonic cell mass shrinks when the trophoectoderm is cut, but it is confirmed that after 48 hours the embryo is still alive. If this method is applied to humans it would require perfusion of the uterus to recover embryos 6-8 days after fertilisation.
There are a variety of methods for preimplantation diagnosis as outlined. This is a method involving the collection of a variety of techniques, including IVF-ET, micromanipulation, genome analysis and genetic disease analysis. However, there are many issues that need to be overcome for application. One is the cost of diagnosis. IVF-ET is very expensive, and individuals cannot afford the total cost, so we need to have some cost coverage to lower the economic burden. We also have to collect as many live ova in good condition as possible. The live birth rate of IVF-ET must be increased, from the current 10% level, which means that 90% of the embryos will be wasted. For mothers who are the subject, the 10% may be a high chance, but the during the operations there may be some damage, so we should increase the efficiency. On the other hand, mothers who use IVF may have some infertility problems from the beginning, so we may expect a higher rate of take home babies. The clinical application of preimplantation diagnosis depends on this.
Finally, I'd like to discuss the ethical issues. Embryo manipulation may damage the embryo, also we need to remove embryos and collect several blastomeres. After IVF on principle we return a maximum of three embryos, the rest are frozen and kept for the next cycle, if needed. As a result of freezing, some blastomeres will die, but after a successful pregnancy a baby is born. Some damage to the blastomeres during these procedures does not give an adverse effect on embryos, and if we remove only 1-2 blastomeres there is no significant effect. However, how do we deal with abnormal embryos? They will be disposed of unless there is some form of treatment available. If we regard the fertilised egg and early embryo as the start of the individual life there are some major ethical issues. There may be genetic treatment for the abnormal embryos in the future, but this type of gene therapy would mean that the genes would be transferred to the reproductive cells, germ-line gene therapy. This would give impact to the next generation. A fourth issue is that we are using embryos, we are trying to do some experiments on very early embryos. Considering that the contraceptive ring which kills the early embryo is used widely, there may not be any legal problems with such experiments.