Preimplantation Genetic Diagnosis (PGD) 
By Dr. William Kearns

Preimplantation genetic diagnosis (PGD) is a state of the art procedure used in conjunction with in vitro fertilization (IVF) to select embryos free of chromosomal abnormalities and specific genetic disorders for transfer to the uterus. These genetic conditions can interfere with embryo implantation, result in pregnancy loss, or in the birth of a child with physical problems, developmental delay or mental retardation.

PGD may be recommended by your physician when there is a possibility, indicated by your medical history or advanced maternal age, that your embryos could be affected by a genetic disease. PGD testing can only be performed within the context of an IVF cycle where eggs and sperm, united in the laboratory, develop into embryos. The Shady Grove Center for Preimplantation Genetics offers PGD screening for chromosome disorders and single gene defects. Genetic testing on sperm from men with male factor infertility or those carrying a balanced translocation is also performed. The Center as well conducts research to evaluate and continually improve the clinical application of PGD and to develop new tests and genetic therapies to benefit a wider variety of patients in the near future.

Indications for PGD

• Recurrent miscarriages
• Unsuccessful IVF cycles
• Unexplained Infertility
• Advanced maternal age
• Male factor Infertility
• Inherited genetic disease

Today, PGD technology reduces the potential for adverse pregnancy for couples ‘at risk’ by enabling us the ability to test the embryos for certain genetic abnormalities before they are chosen for transfer back to the woman. For example: 10 embryos resulted from an IVF cycle and through PGD testing, 6 were identified as genetically abnormal and four were normal. Armed with this knowledge, only the normal embryos would be selected for embryo transfer thereby reducing the possibility of miscarriage or birth defects.

Infertility and Repeated Miscarriage
PGD is recommended most frequently for patients with unexplained infertility, recurrent miscarriages, unsuccessful IVF cycles, advanced maternal age, or male factor infertility. The most likely cause is a chromosome abnormality. Chromosome abnormalities include aneuploidy and structural abnormalities. Anueploidy is the most common chromosomal abnormality. Aneuploidy can occur in both eggs and sperm. Structural abnormalities include translocations, inversions, and deletions. Structural chromosome abnormalities can also be present in eggs and sperm. The transmission of a chromosome abnormality to an embryo can result in a low implantation rate, miscarriage or the birth of a baby with a genetic disorder. Using Fluorescence in situ Hybridization (FISH), the scientists in our PGD laboratory can identify each normal developing embryo for the absence of these specific genetic disorders. As a result, only those embryos free of genetic disease will be transferred to the patient’s uterus so as to increase the chance of conception and ultimately a healthy baby.

Recurrent Miscarriage 
Fertile couples with repeated miscarriages should be evaluated for the presence of a chromosome abnormality. The female or male partner may be a carrier of a balanced translocation or be an aneuploid mosaic.

Unsuccessful IVF cycles
Couples with repeated unsuccessful IVF cycles should be evaluated for the presence of a chromosome abnormality. The female or male partner may be a carrier of a balanced translocation or be an aneuploid mosaic.

Unexplained Infertility 
The most probable cause of women with a history of infertility or habitual miscarriage is a chromosome abnormality. The male or female partner may be a carrier of a translocation or be an aneuploid mosaic. 

Anueploidy and Advanced Maternal Age 
Women of advanced maternal age (> 35) are at a higher risk of producing aneuploid embryos resulting in implantation failure, a higher risk of miscarriage or the birth of a child with a chromosome abnormality (i.e. Down syndrome). This is due to the fact that all of the woman’s eggs are present at birth. Over time, the chromosomes within the egg are less likely to divide properly resulting in cells with too many or too few chromosomes. Aneuploidy is also believed to be a major reason why fertility decreases with age. Prior to attempting a pregnancy, women in this age group may wish to talk with their physician or a medical geneticist about their chances of having a child with a genetic disease and how PGD may be able to help.

Male Factor Infertility 
Approximately one-half of all infertility is caused by sperm abnormalities. Many sperm disorders are due to a chromosome abnormality such as aneuploidy or a structural chromosome abnormality. Men carrying a balanced translocation chromosome are at risk of producing sperm with a structural chromosome abnormality. Dr. Kearns’s laboratory has shown that approximately 3 to 8% of sperm from normal, fertile men are aneuploid. In contrast, between 27 and 74% of sperm from men with severe infertility (i.e. low sperm count, poor morphology, poor motility) is aneuploid. Couples with infertility due to male factor, should consider chromosome analysis on the males sperm prior to IVF.

Y Chromosome Deletions and Infertility 
Y chromosome deletions are found in approximately 5 to 20% of males with a very low sperm count. These deletions appear to impair normal sperm development. While these deletions do not appear to cause any genetic disease, they appear to decrease the chance of men with a low sperm count to successfully fertilize eggs in a normal way.

Genetic Causes 

Abnormal Chromosome Number: Aneuploidy 
The most common type of chromosome abnormality is having to many or to few chromosomes. This is called aneuploidy. Aneuploidy is always associated with physical and/or mental developmental problems. The condition at birth is directly related to the type of chromosome abnormality present in the embryo at the time of conception.

Having an extra chromosome is called trisomy and missing a chromosome is monosomy. If the extra or missing chromosome is an autosome (chromosomes 1 to 22), the embryo may not implant or may stop normal development soon after attaching and undergo a spontaneous abortion. If the aneuploidy involves chromosomes 13, 18, 21 X or Y, the embryo may implant and carry to term. Down syndrome (trisomy 21) is the presence of three copies of chromosome 21.

Figure: Fluorescence in situ hybridization

Patau syndrome (trisomy 13) is the presence of three copies of chromosome 13. Edward syndrome (trisomy 18) has three copies of chromosome 18. Other common aneuploidies seen at birth include Klinefelter syndrome and Turner syndrome. Klinefelter syndrome is the presence of an extra sex chromosome (47,XXY), whereas Turner syndrome is missing a sex chromosome (45,X). Embryos affected with Klinefelter syndrome or Turner syndrome may also spontaneously abort.

Structural Chromosome Abnormalities 

Translocations 
There are two types of structural chromosome aberrations, Robertsonian and reciprocal translocations. Translocations occur when pieces of a chromosome are attached to the wrong chromosome. 

Robertsonian Translocations 
Robertsonian translocations are the joining together of chromosomes 13, 14, 15, 21 or 22. People with a Robertsonian translocation are normal because they have the correct amount of genetic material (genes). Sperm and eggs from individuals carrying a Robertsonian translocation either contain the correct amount of genetic material (be balanced) or contain an unbalanced amount of genetic material (unbalanced). If sperm or an egg contains an unbalanced amount of genetic material and fertilization occurs, the resulting embryo will have too many copies or parts of one chromosome and too few copies or parts of the other. This results in too many or too few normal genes on a chromosome. An unbalanced state in an embryo may lead to embryo death, miscarriage, or the live birth of an infant with substantial medical problems.

• Uniparental Disomy (UPD) and Robertsonian Translocations Genomic imprinting is defined as the differential expression of genes based on their parent of origin. Imprinting plays an important role in early development. Disrupted imprinting can give rise to birth defects. The fetus can have physical abnormalities and intrauterine growth retardation. Since embryos with UPD of some chromosomes are at risk for medical complications, UPD testing may be considered. Testing can be done for UPD by comparing the DNA from each parent to the DNA of the embryo.

Reciprocal Translocations 
Reciprocal translocations are the exchange of chromosomal material between the wrong chromosomes. If this exchange breaks a gene, this person will have a genetic disease. However, if the amount of genetic material present is the same as with normal individuals, the person is balanced and normal. However, the sperm or eggs of these individuals can carry the reciprocal translocation chromosome and are at increased risk of producing an embryo with an abnormal amount of genetic material (be unbalanced). As with Robertsonian translocations, the couple is at increased risk for repeated miscarriages, repeated unsuccessful IVF cycles or the birth of a child with a genetic disorder.

Chromosome Deletions
Deletions are the loss of a chromosome segment resulting in an imbalance in the number of genes present. If the deletion removes genetic material, the individual will have a genetic disorder. Cri du Chat, Prader-Willi and Angelman’s syndrome are examples of genetic disease caused by a chromosome deletion.

Chromosome Inversions 
Inversions occur when a single chromosome breaks in two places and the material in-between is reconstituted upside down. If the chromosome breaks and does not disrupt any gene, people with an inversion are normal. However, if a gene sequence is altered, the individual will have a genetic abnormality. One inversion on chromosome 16 may cause a type of leukemia. The presence of an inversion chromosome during egg or sperm development can result in gametes with a duplicated and/or deleted portion of the inversion chromosome. This is considered an unbalanced state. Embryos with too many or too few copies of genes from this inverted chromosome can fail to grow, miscarriage or be liveborn with substantial medical problems.

Inherited Genetic Disease 
Single gene defects are caused by single gene abnormalities. This is called a mutation. The mutation may be present on a single chromosome of a pair or on both chromosomes of the pair. In some cases the mutation can be in mitochondrial DNA. Mutations cause an error in the genetic information of the gene that alters the normal function of a cell. Gene mutations can alter the cells function due to a lack of a required protein. Single gene disorders usually show a characteristic family history of a specific genetic disease. Examples include Cystic fibrosis, Sickle cell disease, Fragile X, Thalassemia, and Duchenne muscular dystrophy.

PGD is recommended for families with a history of a specific genetic disease. Using polymerase chain reaction, fluorescent PCR and DNA sequencing, scientists can identify each normal developing embryo for the absence of these specific genetic disorders. As a result, only those embryos free of genetic disease will be transferred to the patient’s uterus so as to increase the chance of conception and ultimately a healthy baby.

Single gene disorders are categorized depending upon whether the gene is located on the X chromosome, an autosome or whether the gene is dominant or recessive. These classifications include autosomal recessive, autosomal dominant and X-linked.

Dominant Disorder 
For a dominant disorder, one only needs to have the abnormal DNA sequence on one chromosome. If that mutation is passed on to the embryo, the embryo will be affected with that genetic disease. One example of an autosomal dominant disorder is Myotonic dystrophy.

Recessive Disorders
Recessive disorders require that the mutation be present on both chromosomes of the chromosome pair. If one only has the mutation on one chromosome, the individual is normal but carries the mutation in his cells and is called a carrier. The fertilization of an egg from carrier parents may result in an embryo having the mutation on both chromosomes of the chromosome pair and the embryo being affected with that genetic disease. For example, Cystic fibrosis (CF) is a common autosomal recessive genetic disorder that primarily affects the lungs of CF patients. The CF mutation affects a protein within the cell that reduces the cells ability to function properly. This results in a build up of mucous within the lungs, lung dysfunction and possible death.

X-linked Disorders 
X-linked disorders are due to mutations of genes on the X chromosome and have different patterns of inheritance due to their transmission on a sex chromosome and whether the embryo is male or female. Examples of X-linked diseases are the Fragile X syndrome and Duchenne muscular dystrophy.

Dr. Kearns is the Director Shady Grove Center 15001 Shady Grove Road Suite 400 Rockville, Maryland 20850