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.
PGS – aneuploidy screening (FISH)
Preimplantation genetic screening (PGS) is a method which enables to find out whether the embryos from in vitro fertilization (IVF) show randomly developed changes in the number of chromosomes.
The assumption is that almost half of the embryos have a chromosome aberration which leads to their early miscarriage. Early miscarriage can be explained as due to a „hormonal disorder“. The frequency of chromosome aberrations in recognized pregnancies is 7,5% at the beginning and only 0,6% at birth. This means that the majority of chromosome aberrations are miscarried in the course of pregnancy. The spectrum of chromosomal defects detected in newborns is due to the fact whether the defect is compatible with intrauterine development.
The most common chromosomal defect diagnosed during pregnancy and at childbirth is Down syndrome – trisomy of chromosome 21 (there are three chromosomes 21 instead of two in the cells). Trisomy of chromosome 18 and 13 is less common. The majority of embryos with those chromosome aberrations are miscarried at an early stage of pregnancy. Other chromosomes which display alterations in their number more often are chromosomes 15, 16, 22 and sex chromosomes X and Y (deviations in number of chromosomes 15, 16 and 22 always lead to a miscarriage).
PGS is offered to couples with a higher risk of numerical chromosome aberrations:
- Couples with recurrent miscarriages up to week 12 of the pregnancy, older women (over 35 years)
- Couples with recurrent failures of IVF cycles
- It should be noted here that the efficiency of PGS which is expressed by the increase in number of children born compared to attempts at in vitro fertilization without PGS has not been reliably proved yet.
PGS is performed by using the FISH method (fluorescence in situ hybridization). In this method short sections of DNA – probes, which are highlighted with fluorescent dyes, attach themselves to the chromosomes. The composition (sequence) of the DNA probe corresponds to the sequence of DNA on the particular chromosome. If the sequence of the probe and DNA of the embryo correspond, the chromosome is permanently highlighted with dye which glows under ultraviolet light. In this way the presence or absence of a particular chromosome can be determined. Usually, aneuploidy of 8 most frequently endangered chromosomes is investigated: 13, 15, 16, 18, 21, 22, X and Y.
Success rate and risks of PGS
Success rate of IVF with PGS (number of pregnancies after embryo transfer in the uterus) is 36% (GENNET, 2007-2012).
Like every method, PGS also has a risk of an „error“, which is indicated around 10% for diagnosis of chromosome aberrations. The risk of an error is given by the fact that the probes are not 100% reliable and that there is so-called mosaicism occurring in embryos – the number of chromosomes in the investigated blastomere does not always have to correspond to the number of chromosomes in the remaining cells of the embryo. Therefore, PGS does not replace prenatal diagnosis in the pregnancy.
It should be noted that only a limited number of chromosomes can be observed in PGD. A child born after a PGD can therefore have a different chromosomal defect or genetic disease which has not been observed by us.
The risk of congenital developmental defects is according to the studies which were conducted so far the same as in the population, namely 3-5%.
Sex selection with X-linked diseases (FISH)
Preimplantation genetic diagnosis with sex selection is offered to couples with genetic disease which is linked to the sex chromosome X when the PGD cannot be performed using the PCR method.
- Sex selection is uniquely performed for genetic reasons.
- Examples of X-linked diseases for which PGD-FISH can be performed are Duchenne/Becker muscular dystrophy, hemophilia, or SCID.
- PGD for X-linked diseases is performed in a similar way as PGS.
Success rate and risks
Success rate of IVF with PGS (number of pregnancies after embryo transfer in the uterus) is around 42% (GENNET, 2007-2012).
If the genetic disease is linked to maternal X chromosome (mother is an unaffected carrier), then 50% of the boys are affected by the disease and 50% of the boys carry the „healthy“ X chromosome. Therefore, the biggest hindrance of the FISH method when performing PGD of X-linked diseases is the fact that by sex selection only we do not recommend 50% of healthy boys for the transfer as well. Therefore, this method is only used in cases when it is not possible to perform a targeted PGD using PCR.
New method Array CGH
We have introduced the screening of aneuploidy using the method Array CGH, which enables us to control 24 chromosomes. The aim of the test is to choose embryos without genetic abnormalities for the transfer in the uterus. Those genetic abnormalities could lead to serious developmental defects and they would also increase the risk of a miscarriage and reduce the chance of a successful pregnancy. Microchips are now used for the screening of the most appropriate embryos – the array CGH method replaced the now obsolete FISH method, which we perform only for sex selection.
The method can be immediately used in a fresh cycle for a three- or five-day-old embryo. However, we mainly try not to use one cell of a three-day-old embryo, but a higher number of cells of a five- or six-days-old blastocyst and perform the analysis only as soon as we have several samples, which is economically more advantageous for the patient.
In addition, cell extraction on day 5 does not practically present any additional risk for the embryo and it has a higher diagnostic value. The embryos are frozen using the modern vitrification method and the transfer of the embryo follows in the next cycle after preimplantation analysis. Besides, we achieve the optimal preparation of the endometrium in this subsequent cycle, which results in a high success rate. This procedure is called PGD dislocation. In this case, vitrification is included in the price, as well as the storage of the material until the transfer.