Center for Human Genetics and Laboratory Diagnostics, Dr. Klein, Dr. Rost and Colleagues

Preimplantation Genetic Diagnosis (PGD)

Dr. rer. nat. Annett Wagner, Dr. rer. nat. Thomas Harasim,
Dr. med. Imma Rost

Scientific Background

Polar body diagnosis (PBD) examines the two polar bodies, formed when the egg after ovulation (first polar body) and after penetration of a sperm (second polar body) reduces the original number of chromosomes from 46 (2 x 23) to 23 due to meiosis. This is necessary because the egg and sperm cell initially carry 46 chromosomes, like all other body cells, which must be halved to one set of chromosomes before fertilization, so that after fusion of the pronuclei from egg and sperm, the original double set of chromosomes is available for the developing embryo. In this way, the embryo receives half of its genetic material from the mother and half from the father.

The examination of genetic material from the polar bodies allows conclusions to be drawn about the maternal genetic material in the egg. An assessment of paternal genetic material is not possible with this method.

Schematic representation of egg maturation and embryo development. Following the first meiosis (meiosis I) the first polar body contains a double set of chromatids (1n2C). The second meiotic division (meiosis II) starts after penetration of the sperm. The 2nd polar body (PK2), contains a single set of chromatids (1n1C). An embryo is formed after fusion of the pronuclei, and mitosis begins. (modified from Wagner et al., Cell Biology 2004)

 

Indication

I. Polar body diagnosis (PBD)

Since in PBD only maternal genetic material is detected, only disorders that can be transferred from the mother can be examined. These are, for example balanced translocations, an exchange of chromosomal material between two (or occasionally more) chromosomes whereby there is neither an increase or a loss of genetic material. A balanced translocation can be further inherited in an unbalanced manner; this means with an increase or a decrease of genetic material. Chromosome disturbances due to unbalanced translocations are usually accompanied by congenital malformations and developmental disorders. As part of in vitro fertilization, the polar bodies of each egg can be examined to determine whether the genetic material within the polar body is balanced or unbalanced. An unbalanced polar body confers an unbalanced status on the egg, which would then not be transferred to the mother. Additionally, inversions (rotation of chromosome parts through 180 ° in the same chromosome) can cause unbalanced inheritance by the offspring and therefore constitute an indication for PBD / Pre-implantation diagnostics (PID).

A second indication for PBD is the exclusion of chromosomal aneuploidy (gain or loss of a whole chromosome). Aneuploidies arise at the start of meiosis of an ovum (occasionally also in sperm). Since the maternal egg cells rest from birth to ovulation at a sensitive stage of cell division, and presumably due to the aging process of the cell division apparatus, these incorrect distributions occur more often with increasing maternal age. It is estimated that about 20% of eggs from 20-25 year old women and over 70% from 35-40 year old women show such incorrect chromosomal distributions. As a result, chromosomal trisomy’s occur more frequently with increasing maternal age, which is why prenatal diagnosis is offered from the age of 35 years.

The most common aneuploidies are the tripling of chromosomes 13, 18 and 21, which lead to the birth of a child with Down syndrome (Trisomy 21), Edwards syndrome (Trisomy 18) and Pätau Syndrome (Trisomy 13). Further frequently occurring trisomy’s (chromosomes 15, 16 and 22) are incompatible with long survival of the embryos; these trisomy’s are commonly found by chromosome analysis following a miscarriage.

In addition to the most common trisomy’s, other incorrect distributions are observed. Consequentially, the following are indications for PBD with an increased risk of a chromosomal imbalance:

  • multiple miscarriages
  • previous chromosome maldistribution in a miscarriage or PBD
  • multiple unsuccessful ICSI/IVF cycles with embryo transfer
  • known translocation in the woman

A further indication for PBD are severe, monogenic inherited disorders which are transferred through the mother. Specifically, these are autosomal dominant diseases whose genes are found on the X chromosome, and thus follow a sex-linked inheritance. As a rule, women with a gene mutation on one of the two X chromosomes do not exhibit the illness, while men or rather boys whose sex chromosomes are made up of one X and one Y chromosome, do. The risk of recurrence for sons of a carrier of an X-linked disease is 50%.

PID is the preferential method for detection of an autosomal recessive disease which only occurs when both parents are healthy carriers of the disease, since with PID both maternal and paternal mutations can be detected.

II. Preimplantation genetic diagnosis (PGD)

Trophoblast biopsy as the basis for PGD captures the non-totipotent cells of the embryonic shell and thus maternal and paternal genetic material. Indications for this examination are therefore:

  • known chromosomal translocation in the partner
  • monogenic disorders inherited through the father
  • autosomal recessive disease

 

PBD

PID

Translocation / inversion

Maternal

+

-

Paternal

-

+

Aneuploidy diagnosis

-    Maternal age

-    Implementation failure

-   Recurrent miscarriage

 

+

-

Monogenic (single gene) disorder

X-linked recessive

+

-

Autosomal recessive

(+)

+

Autosomal dominant

Mother carrier

+

-

Autosomal dominant

Father carrier

-

+


Indications for PBD/PGD

 

Methods

The diagnostic difficulty of PBD and PID is that the examinations are only available for single cells (polar body 1 and 2, trophoblast cells).  This means that the findings cannot be checked with a second sample or more cells.  Since the diagnostic confidence in these systems, despite due care, is not 100%, it is recommended that further prenatal diagnosis by chorionic villus sampling or amniocentesis, be performed following PBD or PID to confirm the results.

 

 

Polar body biopsy: After opening the Zona pellucida using a laser, the two polar bodies are extracted with a pipette (right).


1. Array-CGH (PBD and PGD)

This technique is based on the measurement of dose differences between genetic material (DNA) in polar bodies or trophoblast cells in comparison to known reference DNA. The method has the advantage that the entire set of chromosomes can be analyzed for imbalances. Thus, for example on one hand, a maternal translocation in a polar body can be evaluated to determine whether a balanced or unbalanced translocation is present, on the other hand, all chromosomes are recorded, so that other aneuploidies can be excluded. The expressiveness of array CGH is significantly higher than classical FISH analysis. The method of the PGD can even be used with paternal translocations. An advantage of array CGH over FISH diagnostics is that time-consuming suitability testing for specific probes for the translocation diagnosis are not necessary in the run-up to PBD/PGD.

Array CGH unbalanced translocation (7;8) in polar body 1 - gain chromosome 8 and additional loss chromosome 12.


2. Molecular genetic testing

For all single gene diseases where the mother or father, or both in the case of autosomal recessive inheritance, are carriers of a mutation in a particular gene, molecular genetic analysis methods are used. Here an individual system must always be established for the respective family. This consists on one hand of the direct detection of known mutations in the parents or one parent, on the other hand, an analysis of so-called markers, distinctive, individually different characteristics in the genetic material in the vicinity of the known mutation(s). By combining these detection methods, the diagnostic accuracy of PBD or PGD increases.

Combination of direct and indirect diagnostic investigation (mutation detection and marker analysis, respectively): father and mother carry a mutation (red) in the same gene (blue). The sick child has inherited the mutation from both genes. Near the mutation individually different DNA characteristics (markers) are determined, which, in the present case allow mother and father to be differentiated. The maternal mutation is linked with the markers A, B, C and D; the paternal mutation with the markers 5, 6, 7 and 8. Unless recombination occurs, i.e. an exchange of identical chromosomes sections with the mutation between two markers, which is unlikely in the markers closely adjacent to the mutation, the presence or absence of the mutation in the embryo can only be deduced by the detection of the marker combination. If in one cell the mutation detection is not possible due to technical reasons, the detection of the adjacent markers can exclude the failure of the detection reaction (allelic drop out, ADO) with high security. The combination of direct and indirect diagnosis increases the diagnostic accuracy of PBD / PGD.

 

Implementation of PBD/PGD

Both techniques require close cooperation between fertility clinic and the implementing human genetic laboratory and are performed only following consultation, and in the case of PGD, only after the process specified in the preimplantation genetic diagnosis law (Präimplantationsgesetz).

For both methods, genetic counseling and a preliminary medical discussion must be conducted in advance, in which it is determined whether PBD or PGD is possible for the trait or disease. For monogenic diseases an individual test system must be set up for every family, which usually takes several months to complete.

PBD and PID are not currently part of standard care, therefore all costs must be paid by the couple. This does not apply to genetic counseling in the run up to a diagnostic investigation; this is one of the standard benefits of statutory health insurance.