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Preimplantation Genetic Diagnosis (PGD)

What is PGD?

PGD (Preimplantation Genetic Diagnosis) is a technique in IVF combined with ICSI, in which the genetic condition of embryos is analyzed, with the purpose of selecting for transfer into the uterus only embryos that are believed to be chromosomally normal (or in the case of PGD for single-gene disorders, do not have recognizable genetic disorders).

The Basic Science of PGD

Chromosomes are threadlike structures composed of DNA and protein found in the nucleus of cells. They contain the genetic material (genes) that determines the individual characteristics of an organism and controls body functions. Normal body cells of humans contain 46 chromosomes (22 matched pairs numbered: 1, 2, 3, 4 ….up to 22 and one pair of sex chromosomes, X and Y). One of each chromosome is received from the egg and the other from the sperm. During the cell cycle, in preparation for cell division, chromosomes replicate so that each daughter cell receives the full amount of genetic material.

What types of genetic conditions can be detected by PGD?

(1) PGD for Chromosomal Abnormalities

Abnormalities in the number or structure of chromosomes may occur at various stages of embryonic development (during the formation of the egg or sperm cells, the fertilization process, or the early cell divisions), all of which can give rise to abnormalities in the individual. Having an extra or missing chromosome(s) (described as aneuploidy) or pieces of chromosomes (unbalanced rearrangements) can result in arrest of embryonic development and failure of implantation and pregnancy, as well as miscarriage and the birth of an abnormal baby (e.g. Down’s syndrome). (See table 1) As women age, their eggs become more susceptible to chromosomal defects and aneuploidy rates increase. Thus, the chance of conceiving a chromosomally abnormal baby increases.

Because PGD for aneuploidy determines the presence or absence of selected chromosomes, it can be used to determine the gender of the embryo (XY indicates male, XX indicates female) and to screen embryos for a number of the most common defects in chromosome number. These include the major trisomies (an extra copy of a chromosome) resulting in genetic conditions like Down’s syndrome (trisomy 21), Edwards syndrome (trisomy 13), Patau syndrome (trisomy 18) and Klinefelter syndrome in males (an extra X chromosome). It is extremely rare for other autosomal (non-sex chromosomes) trisomies to survive to or near to term, although they are very common in miscarrying pregnancies (1% - 3% of recognized conceptions). Embryos missing a chromosome (monosomies) generally end in arrested growth either prior to implantation or during the early embryonic stages, and are not found in first trimester miscarriages (with the exception of monosomy 21 and monosomy X, Turner syndrome).

The techniques utilized in PGD of aneuploidy also have the capability of screening for certain unique, specific chromosome rearrangements carried in some families (known as translocations). They do not, however, detect genetic disease or predict congenital malformation.

(2) PGD for Specific Genetic Disorders

PGD has also been successfully applied for testing for single-gene disorders in couples at risk of transmitting a genetic disease. The range of inherited diseases for which PGD has been applied in the last decade has extended to over 100 different conditions, with the most frequent ones being cystic fibrosis (CF) and hemoglobin disorders. Knowledge of the specific DNA sequence involved in the gene mutation is necessary; thus for each couple, preparatory work is required.

More than half of PGD cases for single-gene disorders have been performed by gender selection for X-linked conditions like hemophilia. In such cases, female embryos are selected for (they may be normal or carriers) and male embryos which have a 50% chance of being affected are discarded.

The Application of PGD in IVF

The aim of PGD is to transfer only chromosomally normal embryos into the uterus. Thus, the risk of giving birth to a child with birth defects or discovering during pregnancy that a fetus is abnormal is decreased.

Some studies have reported that PGD increases IVF pregnancy rates and reduces miscarriage rates by eliminating the chromosomally abnormal embryos that are prone to arrest during the pre-implantation stages and in early pregnancy.

Relationship Between Embryo Morphology and Chromosome Abnormalities

Around 50% of all embryos from couples undergoing IVF for infertility (including embryos from young egg donors) are chromosomally abnormal. Although the incidence of aneuploidy is higher in embryos of older women, chromosome abnormalities arising after fertilization occur at equivalent rates in all age groups (about 33% of embryos).

Chromosome abnormalities cannot cause embryos to appear abnormal between the 2 and 8-cell stage because the embryonic genes are not yet expressed. Thus, there is no direct relationship between cleavage stage morphology and aneuploidy.

Studies have shown that poor quality embryos (i.e. those that are fragmented, multinucleated, have irregular sized cells or slow cell division), tend to have higher rates of chromosome abnormalities and decreased implantation potential. The proportion of morphologically normal embryos with chromosome abnormalities has been estimated at around 20% (that varies depending on age of female patient, 16% in 20-34y, 37% in 35-39y and 53% in ≥40y), and in arrested embryos that fail to reach the blastocyst stage, as high as 70%.

Since chromosomally abnormal embryos such as trisomies and those classified as polyploid (> 2 sets of chromosomes) can reach the blastocyst stage and beyond, morphological evaluation of embryos alone is not sufficient to screen out chromosome abnormalities (see Table 2).

IVF Patients for whom PGD May be Appropriate

Several groups of IVF patients may benefit from performing PGD:

1. Women of older childbearing age (>35y) undergoing IVF with their own eggs (see table 3). Aneuploidy is a major factor contributing to low implantation and high miscarraige rates in women of advanced maternal age.

2. Carriers of a balanced rearrangement (translocation) who typically have a high risk of producing unbalanced embryos, particularly in the context of poor reproductive history. Miscarriage rates are generally much higher (20-50%) compared to the background population rate of 15%.

3. Couples who are chromosomally normal but have a history of unexplained repeated miscarriages.

4. Couples who have had repeated IVF failures.

5. Patients using a first time egg donor or an egg donor that has a history of poor embryo quality. A recent study indicates that on average only 43% of embryos from young egg donors are chromosomally normal.

6. Couples at risk of having a baby with a genetic disorder.

How is PGD performed?

Step 1: Ovarian Hyperstimulation and IVF

Females undergo hormonal treatment to stimulate the development of several ovarian follicles. The eggs are retrieved under ultrasound guidance and transferred to the IVF laboratory, where they are inseminated either by addition of sperm to eggs in a dish (routine IVF) or by injection of a single sperm (ICSI). Fertilized eggs are cultured for several days, during which time they undergo cell divisions to form embryos. After culturing for 3 days, the embryos have divided to around the 8-cell stage and are suitable for genetic testing.

Step 2: Embryo Biopsy

An embryo biopsy procedure is performed in which an opening is made in the shell surrounding the embryo, and a single cell (or at the most 2 cells) is removed from the embryo by aspiration into a pipette. This procedure is usually performed when the embryo has between 6- 10 cells. The embryo is placed back into the incubator and cultured for 1-2 days while the cell is analyzed.

Step 3: Analysis

PGD for aneuploidy
The biopsied cell is fixed onto a glass slide and the chromosomes analyzed using a technique called fluorescent in-situ hybridization (FISH). This technique uses different colored fluorescent probes (pieces of DNA that bind to specific chromosomes, serving as markers of those particular chromosomes).

The assessment of aneuploidy in the early embryo is based on analysis of a limited number of chromosomes, although any of the 23 chromosomes can be involved in aneuploidy. The standard panel (5-color FISH) screens for chromosomes 13, 18, 21, X and Y, and the 10-color FISH procedure, which involves a second round of hybridization, includes chromosomes 8, 9, 15, 16 and 22. These particular chromosomes are selected based on the chromosome-specific aneuploidy rates in cleavage-stage embryos and the likelihood of survival of affected embryos to term (see table 4). Studies have shown that screening for these chromosomes detects at least 85% of all chromosome abnormalities because these are the ones most involved in aneuploidy, and also the other chromosome aneuploidies tend to occur simultaneously with these.

Once the FISH procedure is complete, the fluorescent signals are counted using a special microscope and the images stored. The cell removed from the embryo is no longer viable and cannot be returned to the embryo.

PGD for single-gene disorders
The biopsied cell is placed into a tube of lysis buffer to release the DNA and a technique known as the polymerase chain reaction (PCR) is applied. PCR allows a single gene to be copied more than a billion times in a few hours. The specific DNA sequence (gene) of interest is amplified to a level at which it can be detected.

Step 4: Selection of Embryos for Transfer into the Uterus

Once PGD results are obtained, patients will be informed of the genetic status of their embryos by their reproductive endocrinologist/ laboratory staff. Only embryos determined to be genetically normal by PGD are selected for transfer into the uterus. Embryo transfer is performed either at the pre-blastocyst (morula stage on day 4) or blastocyst stage (on day 5 or 6). Embryos are graded morphologically and the best quality embryos are selected for transfer (usually two). Excess healthy embryos are cryopreserved.

How safe is PGD?

PGD is a highly technical procedure requiring a high level of expertise by laboratory personnel performing each step of the procedure. At Reproductive Sciences Center, the laboratory staff performing the biopsy and fixation procedures have extensive experience in micromanipulation and cytogenetic techniques, and the embryo damage rate is minimal (<1%).

The embryo biopsy procedure has the potential to damage the embryo such that it would cease to develop and fail to implant into the uterus. After 13 years of PGD experience around the world, there is currently no evidence to suggest that removal of one or two cells from an embryo around the 8-cell stage negatively impacts implantation. In fact, selection of chromosomally normal embryos via PGD appears to more than compensate for any detrimental affects of the biopsy procedure on implantation rates. Following the biopsy procedure, embryos may show slightly delayed cell division for a few hours, but they then continue to undergo normal development,

As of 2004, over 1000 healthy children have been born from PGD with no reported increase in congenital abnormalities over the general population (3-5%). Despite these encouraging results, it is important to emphasize that PGD is still a relatively new procedure, and long-term follow-up of these children is needed to assure the procedure’s safety.

What are the Advantages of PGD?

PGD aims to lower the risk of giving birth to a child with a genetic abnormality. Since PGD is performed before embryos are implanted into the uterus, it can lower the risk of discovering during a pregnancy that the fetus is abnormal, and thus reduces the likelihood that a couple has to face the difficult decision of whether to continue that pregnancy.

Since only embryos determined to be genetically normal are selected for transfer into the uterus, these embryos may have a higher probability of establishing a successful pregnancy than unscreened embryos. This allows patients to choose to transfer only one or two embryos into the uterus, which minimizes the risk of a multiple pregnancy (associated with assisted reproductive techniques).

The risk of miscarriage may also be reduced with PGD, since many of the abnormalities that result in miscarriage are screened out before embryo transfer. Of all recognized pregnancies, about 10-15% end in clinical miscarriage (in women 35y or older as many as 35% miscarry), and about 50% of abortuses are shown to have a chromosomal abnormality (see Table 5).

In addition, PGD provides the option of gender selection for family balancing.

What Are The Disadvantages and Limitations of PGD for Aneuploidy?

Clinical misdiagnosis, which is the occurrence of a fetus or baby with chromosome abnormalities after PGD, is a risk. Analytical errors may arise from a problem with fixation of the cell nucleus, or a problem with the FISH procedure (e.g. failure of the probes to label the chromosomes, or incorrect interpretation of the fluorescent signals caused by overlapping, split or lost signals).

Another pitfall associated with PGD is a condition called mosaicism in which chromosome anomalies are present in a cell yet not in other cells of that embryo (i.e. cells are chromosomally different within a single embryo). Mosaicism is common in human embryos generated in vitro and contributes significantly to the PGD error rate. Mosaic embryos can develop to blastocysts in culture, however there is a tendency for these embryos to arrest development, particularly if the mosaicism is extensive and chaotic (that is, different cells have different aneuploidies). Mosaicism may be patient-specific, i.e. some patients are predisposed to embryos with this condition, particularly translocation carriers and severe male factors.

Overall, misdiagnoses are believed to occur in about 10% of embryos. There is also a 5-10 % risk that a cell will be non-analyzable or only partially analyzable. Embryos that have no PGD result may be transferred if they demonstrate normal development but the benefits of PGD will not apply.

PGD does not test for the presence of every chromosome, thus some abnormalities of chromosome number or de novo chromosome rearrangements may not be detected. However, aneuploidies for the chromosomes that are not analyzed by PGD are rarely compatible with life, thus embryos carrying these abnormalities are rarely able to establish a pregnancy.

Recommendations for Follow-Up

Due to the possibility of misdiagnosis with PGD, we strongly recommend that you have standard prenatal testing, either by chorionic villus sampling (CVS) or by amniocentesis, between 10-16 weeks to confirm normal fetal development.

What are the limitations of PGD for Single-Gene Disorders?

PGD for the presence of familial single gene defects is a highly complex analysis which requires the sequencing of very small segments of DNA using polymerase chain reaction (PCR). It is possible to misdiagnose an affected embryo as one which is merely a carrier for the disease (like its parents) so that patients who choose PGD for this reason are advised to follow up with prenatal genetic testing by amniocentesis or chorionic villus sampling.

Is PGD Right For Me?

In addition to the indications listed above, other factors may also make PGD a desirable choice, and should be discussed with your reproductive endocrinologist. If you feel that PGD may assist you in your effort to have a healthy child, we invite you to contact us for a comprehensive consultation.

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Tables

Table 1: Frequency of Chromosome Abnormality in Newborns

Abnormality	Chromosome	Rate per 1000
Autosomal trisomy (3 of a particular chromosome)	13	0.08
(3 of a particular chromosome)	18	0.15
	21	1.2
	Total (13+18+21)	1.4
Triploidy		0.02
Sex Chromosome Abnormalities	XXY	1.2
	XYY	1.2
	45, X	0.3
	XXX	1.1
Structural rearrangement, unbalanced		4
Structural rearrangement, balanced		4.3
Overall, babies with a chromosomal abnormality		1 in 120 live born infants

Sources: Hook (1992) in “Prenatal diagnosis and Screening” (D.J.H. Brock, C.H. Rodeck, and M.A. Ferguson-Smith eds.) and Jacobs et al (1992) J. Med. Genet. 29, 103-108, and National prevalence estimates (1999-2001) Morbidity and Mortality Weekly Report, Dec 2005, CDC.

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Table 2. Survival of Chromosomally Abnormal Embryos to the Blastocyst Stage

Abnormality	Blastocyst rate
Trisomies	35-37%
Monosomies (X and 21 only)	9-20%
Haploid	0-10%
Limited Aneuploid 2n Mosaics (< 38% abnormal cells)	17%
Extensive Aneuploid Mosaics (> 38% abnormal cells)	0-6%
Polyploid	21%

Source: Sandalinas, M. (2001) Hum. Reprod. Vol. 16, no. 9 pp.1954-1958

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Table 3: Improvement in IVF outcome using PGD for Advanced Maternal Age.

Age	Implantation rate No PGD, %	Implantation rate After PGD (%)	Improvement due to PGD (%)
35- 37	26	31	+5
37- 39	19	22	+3
39- 42	13	20	+7
42- 45	3	11	+8
39- 45	11	18	+7

Source: Munné et al 2002, Fertil. Steril. 78, 234-236

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Table 4. Chromosome Specific Aneuploidy Rates in Cleavage-Stage Embryos

Chromosome	Aneuploidy Rate, %
XY	1.2
1	2.5
2	0.6
3	2.5
4	2
9	1.2
11	3.7
13	2.9
14	1.1
15	5.0
16	5.3
17	2.5
18	2.3
21	4.9
22	6.2

Note: Average maternal age:37 yr.
Source: Munne, S. (2006) Reprod. Biomed. Online (Feb. 2006)

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Table 5. Findings in Chromosomally Abnormal Spontaneous Abortions

Abnormality	Rate
Autosomal trisomy	60%
Trisomy 15	14%
Trisomy 16	15%
Trisomy 21	8%
Monosomy X (45,XO)	20%
Triploid	15%

Source: Kajii, T. (1980) Hum Genet. 55, 87-98