Preimplantation Genetic Diagnosis (PGD)

With recent advances in genetics, there are several inherited disorders which can now be diagnosed at a molecular level. For couples who are carriers or affected by any of these conditions and are at high risk for transmitting it to their offspring, it is currently possible to detect the disorder during pregnancy. This is done by one of two approaches: amniocentesis or chorionic villus sampling. However the couples have the dilemma of whether or not to terminate the pregnancy if the genetic abnormality is present. In some cases this may also not be a viable option for religious or moral reasons.

An alternative would then be to diagnose the condition in embryos before the pregnancy is established. Only the unaffected embryos would then be transferred to the uterus. This technique is referred to as Preimplantation Genetic Diagnosis (PGD).

Preimplantation Genetic Diagnosis (PGD) is, presently, a valid alternative for families at a high risk for producing offspring with genetic disorders and to those who wish to avoid elective pregnancy termination or to prevent the birth of an affected child following prenatal diagnosis. PGD allows genetic analysis to be performed on early embryos prior to implantation and pregnancy. This provides couples at risk the opportunity to know that any pregnancy they achieve should be unaffected and obviates the need for screening during a pregnancy and hence prevent the physical and psychological trauma, and ethical-moral problems associated with possible termination.

Technical advances in molecular genetics now enable physicians and scientists to be able to diagnose some inherited genetic or chromosomal disorders from a single cell of an early embryo. The information gained by PGD is used to select for replacement in the uterus only those embryos considered unlikely to be affected by the specific genetic disorder for which testing is performed.

Couples who have PGD will undergo an in vitro fertilization (IVF) cycle for the purpose of creating embryos from the woman's eggs and man's sperm which will have genetic testing prior to replacement into the woman's uterus. The genetic material of the embryos (which is derived from both parents) is not altered in any way during a PGD cycle, and early embryological development is similar to natural conception, except that it occurs in the laboratory.

Embryos that show normal development are biopsied with micromanipulation techniques to obtain sufficient cells (blastomeres) for analysis. The cells removed from each individual embryo are analyzed by genetic testing using PCR-based DNA amplification. Those embryos considered to be unaffected on the basis of this testing will then be available to be transferred into the woman's uterus.

The intrinsic difficulties of this diagnostic approach (single cell DNA analysis, response time of 24 hours, the possibility of Allele Drop-Out - ADO), require the use of extremely sensitive and automated techniques that provide unambiguous and reliable results. In performing of PGD cases, “Genoma”� Laboratory uses the most modern and technologically advanced instruments presently available and employs sophisticated and innovative analytical procedures. Avant-garde technologies and robotic solutions applied to the entire analytic process contribute to raise the quality of the analysis, guaranteeing maximum reliability of the results.

These characteristics have enabled “Genoma”� to become one of the most highly qualified European laboratory in the field of preimplantation genetic diagnosis, a reference center for various national and international IVF centers.

To date, “Genoma”� Laboratory has performed over 80 PGD cases, collaborating intensely with prestigious international institutes.

Scientific activity of the center results in the publication of different manuscript in prestigious specialized journals as well as in oral communications in different meeting, among which the 3^rd and 4^th International Symposiums on Preimplantation Genetics.

Preimplantation Genetic Diagnostic procedures have been attempted by GENOMA for various single gene disorders including:

•  Cystic Fibrosis 

• Beta Thalassemia  

• Hemophilia A  

• Retinoblastoma  

• Myotonic Dystrophy  

• Duchenne-Becker Dystrophy  

• Spinal Muscular Atrophy (SMA)   

• Limb-Girdle Muscular Dystrophy

 Research and Development Section of “Genoma”� Laboratory is able to set-up and perform PGD for any genetic disorders with an identifiable mutation.

What are the pathologies that can be diagnosed by PGD DNA analysis?

Almost all genetically inherited conditions that are diagnosed in the prenatal period can also be detected in the preimplantation period. Diseases which have a high risk of transmission (25-50%) and are usually associated with significant morbidity and mortality can be screened for by this technique.

It is possible to perform PGD for any genetic disorders, autosomal dominant, recessive or X-linked, with an identifiable mutation. For pathologies caused by expansions of nucleotidic triplettes (such as Fragile X, Huntington's disease, Myotonic Dystrophy, etc.) it is possible to obtain only information on the absence of triplette expansion. Whether or not to undergo examination must therefore be evaluated case-by-case.  Below is a table listing of the most frequent genetic diseases that can be diagnosed by PGD.

 Table 1. Genetic diseases transmittable to offspring that can be analyzed by genetic diagnosis after biopsy of the ovocytes and embryos:

How is PGD carried out?

PGD involves genetic testing of the oocytes and/or embryos obtained by undergoing IVF.  IVF is an assisted reproductive procedure where fertilization of the egg occurs outside of the body in a controlled setting.  The oocyte (egg) is removed from the woman’s ovary and is placed with the male’s sperm.  If the sperm fertilizes the egg, the fertilized egg (zygote) begins to divide. The genetic status of the embryo(s) can be determined before the embryo(s) is/are transferred into the uterus.  

The following procedures are necessary when undergoing PGD:

•  Ovulation Induction

• Oocyte Aspiration

• Fertilization and Embryo Culture

• Polar body removal and/or blastomere biopsy

• Genetic testing

• Embryo transfer and implantation

 Ovulation Induction

The first step involves stimulation of the ovaries for ovulation. Ovulation is the release of a mature egg from its follicle in the ovary. Most patients who undergo cycles of assisted conception do not have ovulation problems. The aim of stimulation is to induce, in the chosen cycle of assisted reproduction, the simultaneous maturation of several follicles in order to have many ovocytes available and possibly many embryos to transfer. In the IVF process, mature eggs must be retrieved before they are ovulated.  Medication is used during a woman’s cycle in order to stimulate the eggs to mature at the correct time. Ultrasound control of the ovary enables us to know when the eggs are fully mature.

Oocyte Aspiration

When the oocytes are ready to be retrieved, another vaginal ultrasound similar to those performed to monitor follicular development is performed. A small needle inserted in a guide attached to the ultrasonic probe is directed through the vaginal wall into the ovarian follicles. The needle is connected to a suction pump and the fluid from each accessible ovarian follicle within the ovary is aspirated. 

Fertilization and Embryo Culture

The laboratory testing procedures take place in a special laboratory where all conditions are sterile.  The egg cell prior to fertilization divides into two unequal cells.  The larger cell is the mature egg that will be fertilized.  The smaller cell (called a polar body) can be removed and tested for its genetic composition by a procedure called polar body removal.

Polar body removal and/or blastomere biopsy

Blastomere Biopsy

Blastomere biopsy (also known as embryo biopsy) is a technique that is performed by removal of one or two cells (blastomeres) from the 4 to 8 cell pre-embryo stage for the purpose of preimplantation analysis. The egg will typically be fertilized by using intracytoplasmic sperm injection or ICSI. Following fertilization, the zygote begins to divide.  On the third day following the egg retrieval, the embryo is at the blastomere stage, and a cell may be carefully removed for genetic analysis.  With the embryo maintained in position by a pipette with rounded margins, an opening is performed  in the embryo by using a laser device or thyroid acid: Once realized the hole, a new micropipette having a greater diameter than the first is positioned: this will consent, by means of aspiration, the removal of a cell(s) that will be then be released by applying negative pressure. At this early point of embryo development, all of the cells are equivalent and thus, removal of a cell from the embryo at this stage does not remove anything critical for normal development.  The embryo compensates for the removed cell and should continue to divide following blastomere biopsy. Therefore, if the technique is carried out correctly, there are no risks for the embryo.

After removal of the cell(s) from the blastomere, the developing embryo is placed back into the culture dish and the removed cell(s) is inserted into a test tube for subsequent genetic analysis.

Polar Body Removal

 As indicated above, the first polar body is produced from the division of the egg and can be removed and tested for its chromosome complement or to identify whether it contains the abnormal gene of concern.  Upon penetration of the egg by the sperm (fertilization), but prior to the joining of  the sperm’s genetic material with the egg’s genetic material, the egg undergoes another cell division, producing two unequally sized cells.  The larger cell will join with the sperm’s genetic material to create the pre-embryo, and the smaller cell is called the second polar body.  The polar bodies have no known function except to assist in cell division.  They are simply “by-products”� of the egg’s division. Once implantation occurs, the polar bodies disintegrate and are not part of the developing fetus. 

By testing the first and second polar bodies, the genetic make-up of the egg, and maternal genetic contribution in the resultant embryo, can be determined. Removal and genetic analysis of the polar bodies occurs on the first and second day after aspiration. In some instances, it is necessary to confirm a diagnosis made on polar body analysis by performing blastomere biopsy.  It is also possible that one or more polar bodies fail to provide a conclusive result.  In these situations, it may be possible to perform blastomere biopsy (embryo biopsy) for further genetic analysis.

 Preimplantation Diagnosis of Single Gene Disorders in blastomeres

Currently, PGD for single gene disorders can be accomplished by either polar body removal or blastomere biopsy.  Which method may be used is determined on a case-by-case basis.  This procedure can be used to select for embryos that do not have a specific genetic disease by testing the polar bodies or blastomeres for the genetic mutation. 

Inside the test tube containing the blastomere, a solution is added that permits cell lysis and thus the liberation of DNA from the cell nucleus. Subsequently, by means of in vitro enymatic ampification known as the Polymerase Chain Reaction (PCR), the genetic region of interest, involved in the mutations that is being searched for, is amplified millions of times. The genic amplification product then undergoes mutation analysis to search for genic mutions present in the couple. The analysis of mutations is the most important and delicate phase of PGD. To guarantee maximum interpretative reliability, it is indispensable that it be carried out with methods and instruments that permit the univocal identification of the mutations that are being searched for. Automated sequence analysis is presently the best method of genetic analysis, inasmuch as it permits the exact determination and direct visualization of a specific mutation. The application of this technique to PGD is done by the use of completely automated state of the art equipment.

PGD centers worldwide are concerned about the possibility of misdiagnosis, which can occur as a result of failure of allele specific amplification, or allele dropout (ADO).  In basic terms, this is the failure of one of the genes (allele) to show up in the analysis (it “drops out”� of the picture.) ADO is of concern primarily in blastomere biopsy when each parent carries a different mutation for a recessive condition.  To minimize the potential for diagnostic error by PGD, it is preferable to offer polar body removal or to perform a combined analysis of the specific mutation and specific linked markers that are inherited along with the gene. This dual amplification allows for higher accuracy and for the detection of ADO, thus enhancing diagnostic accuracy and reducing the risk for misdiagnosis.  

Embryo transfer and implantation

After mutation analysis of the blastomeres has been completed, once an embryo is predicted to be free from the genetic disease for which testing was performed, embryos resulted without genetic anomalies are transferred to the patient.

Embryo transfer is a 15 minute procedure accomplished by inserting a catheter (preloaded with embryos) into the uterine cavity through the cervical canal.  This procedure is often performed under ultrasound guidance.