With a fast increase in the quantity of known facts about the human embryonic development under the condition in vitro, the methods of assisted reproduction have undergone a huge extension in recent years. Thus it is no wonder that methods of early detection of chromosomal deviations, or early prenatal diagnostics, are also being developed at the same time. One of the methods of an early prenatal diagnosis, which has been applied in this branch, is the preimplantation genetic diagnosis (further PGD).
PGD makes it possible to reduce the risk of spontaneous abortions by means of a selection and transfer of the embryos, which are not affected by any severe chromosomal aberration or another genetic disturbance, whose consequence is an early resorption of the embryos or a selective abortion. Thus the parents may start the pregnancy with a feeling that their future descendant will not suffer from any severe genetic condition or a defect occurring in the family, which can be examined by the methods of prenatal genetic diagnostics.
Principle of PGD
Before the couple may undergo the preimplantation genetic examination, they are sent for an interview with a clinical genetic expert. This doctor will perform the appropriate genetic examination (especially the examination of caryotype from peripheral blood), which makes it possible to determine the risk rate of a genetic burden of the couple and recommends the optimal procedure. If the appropriateness of the PGD is concluded, the couple will undergo the IVF cycle. On the third day after the fertilisation, when the embryos achieve a stage of eight cells, the embryologist carefully collects 1 – 2 cells from every embryo. These cells undergo further steps of the genetic examination. Within this developmental stage the embryo rapidly replaces the collected cells without any damage and continues further in its development.
The result of the genetic examination is the determination of which of the embryos are, in terms of the selected genetic analysis, the healthy ones. Only these embryos are introduced into the patient’s uterus on the fourth to fifth day. The total period, which is spent by the examined embryo under in vitro conditions is the same as in the case of non-examined embryos.
General indication to PGD:
Couples with a carrier of a chromosomal aberration
Couples with the occurrence of a hereditary disease related to the chromosome X.
Couples with a carrier of the so-called small chromosomal mosaics.
Couples with repeated reproduction losses.
Couples with repeated failure of the IVF cycle.
Couples, where the testicular or epididymal spermatides (obtained with the method MESA or TESE) were utilized for the ovum fecundation.
Couples after an undergone chemo-/radiotherapy.
Couples with a genetic disease in the family (some monogenous diseases).
Couples of a higher age (especially if the woman’s age is over 35 years).
Advantages and disadvantages of PGD
In case of the molecular cytogenetic examination it is necessary to take into account the possibility of an error as a consequence of the chromosomal mosaicism, when a proportion of the embryonic cells is normal while the other cells contain a pathological chromosomal feature. Up to 50 % of embryos may be disunited in their chromosomal equipment. For any further embryonic development the percentage representation of normal and pathological chromosomal equipment is important. It has been demonstrated that a small pathological clone is able to eliminate the embryo during the further development.
Thus the embryo could develop into a foetus with a fully normal chromosomal equipment, however, during the preimplantation genetic examination, it could be characterised as a pathologic one. The risk of this erroneous diagnosis is about 1.3 %. The opposite situation can also arise. The embryo could be denoted as a normal one, however, its chromosomal equipment would be pathologic. The risk of this error is about 4.3 %.
In this context we cannot miss a special group, which is formed by embryos with fully chaotic genetic equipment. Each of the cells of this so-called chaotic embryo carries a different chromosomal aberration. As a different type there are embryos with tetraploid or octaploid number of chromosomes.
The increased representation of embryos with abnormal chromosomal equipment – aneuploid equipment, mosaics, chaotic caryotype – is found in infertile couples.
In case of carriers of structural chromosomal aberrations it is possible, with the help of PGD, to mark the embryos with a chromosomal imbalance. However, it is not possible to safely distinguish the completely sound embryos from the ones being relatively healthy but carrying the genetic aberration, as one of the parents. The carriers of structural chromosomal aberrations (for example, translocations) are, in addition to being affected by the creation of genetically imbalanced gonadal cells due to the given chromosomal aberration, also burdened by the so-called interchromosomal influence of their chromosomal aberration to other chromosomes, which can lead to the origin of further chromosomal defects (especially aneuploidy of other chromosomes).
In case of PGD, therefore, it is necessary to count with the risk, that the whole IVF cycle may not be completed with an embryo transfer into the uterus. The examination may be foiled by a low number of cleavaging embryos, incorrect manipulation of the collected embryonic cell or a contradictory result of the PGD. As it was mentioned above, the genetic analysis may give incorrect results.
The PGD examination is always focused only on the selected type of chromosomal defect and it is not excluded, that in case of pregnancy a suspicion for another genetic defect will be expressed.
Although PGD cannot fully substitute the genetic examination of the foetus at a later stage of pregnancy (for example amniocentesis), it increases the chances of successful implantation of the embryo into the uterus, reduces the risk of spontanneous abortions at a later stage, and particularly significantly reduces the risk of genetic affection of the foetus.
Laboratory methods of PGD
The cells collected form the three-day-old embryo are most often tested using the method of fluorescent in situ hybridization (FISH). This molecular cytogenetic technique is suitable in case of an examination of aneuploidy of the embryos, determination of sex of the embryos or identification of the carrier of the structural aberrations. For the detection of a monogenous disease a genetic method known as the polymerase chain reaction (PCR) is used.
The fluorescent in situ hybridization is based on the principle of hybridization of a short fluorescently labelled sequence of DNA (a so-called probe) with the corresponding sections of the target DNA sequence from the embryonic cells being examined. This hybridization result is deducted from with the assistance of a fluorescent microscope. Thus we obtain an overview of the number of copies of the selected section of DNA, representing a certain chromosome, in the nucleus of the examined embryonic cell.
The polymerase chain reaction was developed in 1980’s and today it represents one of the most common tools of the molecular genetic diagnostics. The basis of this highly efficient method is in vitro synthesis of a selected section of DNA, which proceeds in many repeated cycles. As a result we obtain a large number of copies of the given section of DNA. This amplified fragment can subsequently be separated and visualized by means of electrophoresis. The most sensitive electrophoresis type is the so-called capillary electrophoresis, which we have for disposal in our laboratory. With the help of these methods it is also possible to analyse a very small quantity of DNA, thus also DNA contained in only one cell.
PGD for carriers of chromosome aberrations (FISH)
The FISH (fluorescent hybridization) method is a more sensitive cytogenetic method. In case of this method the chromosomes are marked with short sections of DNA – probes – labelled with fluorescent agents. The DNA sequence of the probe is complementary to the sequence of the DNA in an accurately determined location of the chromosome. If the sequence of the probe and the DNA of the patient correspond to each other, the chromosome is permanently labelled by the colour agent, which shines in the UV-light. Thus it is possible to demonstrate the presence or the alteration of a certain gene. The green signal on the picture denotes the centre (centromere) of the chromosome and the two red dots represent the ends of the short arms of the chromosome X.
The FISH method is often utilized in preimplantation diagnosis (PGD) as part of the assisted reproduction (IVF), before the embryo is inserted into the uterus. Most frequently, 1 to 2 cells are taken from the embryo, the centres (centromers) of selected chromosomes are marked with fluorescently-labelled probes and thus their number is determined. Thus it is, for example, possible to find out, whether the embryonic cells contain three chromosomes No. 21, which would cause the Down syndrome. As well as that, it is possible to determine the sex of the embryo.
Chromosomal PGD may increase the hope of success of the assisted reproduction, mainly in case of older mothers and the pairs with a repeated non-success of the IVF.