pp. 429-431 in Bioethics in Asia

Editors: Norio Fujiki and Darryl R. J. Macer, Ph.D.
Eubios Ethics Institute

Copyright 2000, Eubios Ethics Institute All commercial rights reserved. This publication may be reproduced for limited educational or academic use, however please enquire with the author.

F25. The Present State of and Issues Concerning Preimplantation Diagnosis

Kaoru Suzumori.

Nagoya Municipal Medical University, Nagoya, Japan

About a quarter of a century has passed since prenatal diagnosis started in Japan with amniocentesis. During this period many improvements have been made in the ultrasound and other equipment used in diagnosis, leading to improvements in image quality. This in turn has led to dramatic advances in the safety and reliability of amniocentesis, and its coming into general and widespread use, so that now amniocentesis not only plays an important part in genetic counselling, but is also established as a screening method for chromosomal abnormalities in pregnancies in the over-35 age group. Looking at how techniques of prenatal diagnosis have changed one can see that there has been a shift to earlier diagnosis with the aim of lightening the physical and mental burden on the woman in abortions of pregnancies based on findings of abnormality, and that a number of institutions have experimented with amniocentesis and chorionic villi sampling early in pregnancy.This has had the result of widening the choice of diagnostic procedures for the testee. Looking at the diagnostic methods for genetic disease, as well as the molecular genetic procedures such as chromosomal analysis and genetic biology techniques such as enzyme analysis used hitherto, recently the method of gene DNA analysis has also come to be used. The application of this particular technique has not just widened the range of target diseases, but also means the sample is not selected, and that diagnosis can be made from a very small amount of test material. It has also led to improvement in diagnostic accuracy. The prenatal diagnostic procedures that are involved in genetic counselling have increased from amniocentesis and chorionic villus sampling as fetal blood sampling and fetal biopsy have become possible, and so not only have the types of genetic disease that are the objects of testing increased even further in variety, but also the situation has been reached where it is possible to select the most appropriate procedure in line with the diagnostic method for the disease, the organ or tissue expressing the abnormality etc.. However these procedures all have as their subject the fetus, and, depending on the outcome of diagnosis can lead to the abandonment of the fetal life itself, or putting it bluntly, abortion of the pregnancy, and so have become an ethical and social issue.

The 1980s were a period of striking advances in reproductive technology during which treatment of infertility by IVF and embryo transfer (ET) became established also in Japan, were it is now carried out at many institutions. Preimplantation diagnosis was born out of a combination of these assisted reproductive technologies and molecular biology (gene DNA analysis , its most distinguishing characteristic being that diagnosis is possible before pregnancy is achieved, so the child can be brought into the world without the fear that it will be aborted. At present it is in actual use at about 36 institutions in the U.S.. In a representative procedure at the 4-8 cell stage of the embryo 1 or 2 blastomeres are biopsied under the microscope, genetic analysis is performed, and a judgement on normalcy made. Only normal embryos are chosen and used to induce pregnancy.

The Principles of Preimplantation Diagnosis

1. As in normal IVF and ET the ovaries are stimulated to superovulation. This is done as the more good quality ova are retrieved and embryo used in the diagnosis, the more normal embryos, returnable to the uterus, can be obtained.

2. Insemination

3. The fertilized egg is placed in a petri dish and cultured in an incubator

4. The embryo is cultured to the 4-8 cell stage by leaving it to stand.

5. 1 or 2 blastomeres are biopsied from the 4-8 cell embryo. While holding one side of the embryo using a micromanipulator and fixing it with a pipette, the sample blastomeres are removed from the other side using a sampling pipette.

6. Culture of the biopsied egg (embryo) is continued until the results are known.

7. Only embryos judged to be normal are returned to the uterus (embryo replacement).

Should pregnancy be achieved , the fetus will be normal. We can say that this method of preimplantation diagnosis is a cutting-edge technology, based on IVF and ET, and only made possible by free use of manipulations developed for microfertilization, and the techniques of molecular biology making possible gene DNA analysis of genetic material from minute amounts of sample.

Molecular Biology-Based Methods of Diagnosis of the Embryo

1. Sex determination of the embryo

Preimplantation diagnosis is a technique arousing medical, bioethical and societal interest, and as you all know the first successful clinical application of this procedure attracted worldwide attention. This first successful case was reported by a group at Hammersmith Hospital in England, the subject being a woman heterozygous for a hemophilia gene. Given the techniques available at the time it was only possible to determine sex. In outline the following was done; for the couple who did not want a sick child, all the early stage embryos determined to be male were disposed of, and only those diagnosed as female were transfered, with the anticipated girl being born. As in this case preimplantation diagnosis started from sex-determination for carriers of X-linked genetic diseases.

At present there are two molecular-biological methods available for sex-determination-PCR (polymerase chain reaction) and FISH (fluorescence in situ hybridization). In PCR the target DNA sequence from a minute amount of sample is amplified 2-5 hundred thousand times and diagnosis made on the presence or absence of PCR product or on the difference between individuals. In the successful example of preimplantation diagnosis just related, this method was used to determine sex based on whether a particular sequence of the Y chromosome, which is specific to the male, had been amplified or not. Specificity is high for the Y chromosome, while if the region is easily amplified the detection sensitivity is also high. However if the genetic material is not well extracted then even a male embryo will be negative for a Y-specific region, that is to say there is the danger it will be diagosed as female. At this stage a system was devised to monitor whether DNA was being extracted or not, at the same time as increasing the certainty of sex determination, by also amplifying an X-chromosome specific region. Using this method, even for a female rather than the sample being totally negative for bands an X-band can be observed, confirming the extraction of DNA, and accuracy is increased markedly over sex-determination based simply on the presence or absence of the single Y-amplified constituent band. The limitation of sex-determination by the PCR method is its non-quantitative nature, the presence or absence of the X and Y chromosome is determined, but not their number. That is to say abnormalities in the number of chromosomes such as X monosomy, XXX and XXY individuals etc. cannot be detected.

The FISH method applies the principle of labelling a DNA clone with a chemical substance such as biotin, and after hybridizing this labelled clone with the chromosome or nuclear DNA, reacting with avidin FITC which has a high affinity for biotin. The FITC is visualized on a fluorescent wavelength enabling the presence and position of the DNA probe to be clarified. If a DNA probe emitting a strong, high-rate signal and chromosomal-specific (_-satellite, cosmid-probe etc.) is used, then based on the presence and number of the DNA probe signals not only sex-determination, but also the on the spot diagnosis of chromosome numerical abnormality is possible. Using this FISH method, if probes for other chromosomes (the clinically significant 13, 18, 21) are used then without being limited to just the X and Y, it is possible to also diagnose numerical abnormalities in these chromosomes at the same time. When applying the FISH method to preimplantation diagnosis whether or not a single cell (blastomere) can be dropped into place on the slide correctly fixed in position is important. It is also necessary to pay the utmost care during the processes of hybridization and lavage etc. as the cell may be torn away. Also due to the thickness of the nucleus itself, on occasions the signals cannot be detected at the same focal point and there exists the danger of overlooking signals that actually exist, leading to mis-diagnosis.

2. Diagnosis of Single-Gene Diseases

Recently gene diagnosis of early embryos has expanded to single-gene diseases, and in 1992 there were successive reports of successful examples of pre-implantation diagnosis of cystic fibrosis and Tays-Sachs disease. Recently I visited Dr. Joyce Harper of University College London and procured the latest summation of results of preimplantation genetic diagnosis. What is clear is that at present in the 36 centers in the world preimplantation diagnosis was being carried out not only for X-linked diseases, but also for cystic fibrosis and Tays-Sachs disease. These diseases occur at a high frequency among Caucasians and Jews, and it seems that the fact that the region of genetic mutation is 80-90% the same makes pre-implantation diagnosis favourable in the U.S. and Europe. Also the fact that for religious reasons some believers cannot choose to have a pregnancy artificially terminated is probably instrumental in heightening the clinical significance of preimplantation diagnosis. Recently the PEP (primer extension amplification) method, by which about 70-80% of the entire genome can be amplified from a individual cell, has been developed, so it has become possible to obtain multiple genetic information from an individual cell. Using the PEP product and amplifying multiple target regions using the PCR method a rather detailed diagnosis is possible. Taking the X-linked disease DMD as an example, and supposing that it has been ascertained beforehand what kind of genetic mutation ( exon deletion etc. ) might be present, first of all the presence or absence of a Y-chromosome is diagnosed using PCR for the purpose of sex-determination. Should the embryo be male, then next the presence of the exon deletion can be investigated, and hence the disease state diagnosed. If this procedure is utilized the tragedy of a normal male child being aborted only on the basis of a sex-determination will disappear, which will be good news for families who want male children.

Preimplantation and Bioethics

New ethical problems arise accompanying the development of fetal medical science and assisted reproductive technologies. The problem arising with prenatal diagnosis was the great debate that took place and is still taking place around the handling of fetuses diagnosed as abnormal, and whether abortion for the reason of abnormality is permissible. The technique of preimplantation diagnosis as I have explained it here is also intimately related to the coming into existence of human life and needs to be looked at in detail from an ethical standpoint.

Morioka classifies the initial and final stages of life into 5 levels according to the nature of the functioning of the central nervous system in each. Preimplantation diagnosis takes place at level 1, the period of 14 days from fertilization. During this time the cells are continuously dividing, while organ differentiation is not yet detectable. The stage of the final period of life equivalent to this level is that at which all bodily functions have ceased, coinciding with a state of physical death. It is at levels 3 and 4 that the presently widely-practised chorionic and amniotic diagnoses are performed, conditions under which the brain, and in particular brain stem, have already started functioning. The state of the final periods of life corresponding to this is the vegative one, and while there is a difference in its level between 3 and 4, these are states where brain function remains. In parliament a resolution has been adopted recognizing brain death as the death of the individual only in the case of organ transplantation, but when we think that the final embryonic and early fetal stages, those at which chorionic and amniotic diagnoses are carried out, are equivalent to the "vegative" state in the final periods of life, logic demands that we consider the fetus during this period to be a " living human being ". Thinking of these problems in this light, it cannot be helped if abortions of abnormal fetuses based on present-day prenatal diagnosis are looked upon as unethical. In Japan as well we need to study the ethical and medical significance of preimplantation diagnosis, and deliberate seriously its place in prenatal treatment.


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