Preimplantation Genetic Diagnosis

Overview

Preimplantation Genetic Diagnosis (PGD) is a technique that provides us with information about an embryo before it is transferred to the uterus (preimplantation). It is different from amniocentesis and chorionic villus sampling which are used to determine fetal abnormalities nine weeks or more after implantation (post-implantation).  PGD technology allows us to identify single gene defects (cystic fibrosis, Huntington’s disease etc), chromosomal translocations (Robertsonian, Reciprocal) as well as numerical chromosome abnormalities (Down syndrome, Turner syndrome) in the early embryo. 

How is PGD Performed?

PGD begins with embryo biopsy

Patients electing to have PGD will undergo an IVF cycle where the woman is given fertility medication to increase the number of eggs that will mature and be retrieved during the egg retrieval.  Her male partner will provide a sperm sample, and the eggs and sperm will either be incubated together to achieve fertilization or each egg will be injected with a single sperm in a procedure called Intracytoplasmic sperm injection (ICSI).  Normally, one-half or more of the eggs will become fertilized and develop into embryos.  The embryos must grow in the laboratory for three days before the PGD process can be initiated.  By this time, the embryos should have between 6 and 8 cells, called blastomeres, and a micromanipulation procedure called embryo biopsy is done.  During embryo biopsy, a small opening is made in the protective coating or shell surrounding the embryo, called the zona pellucida.  A tiny glass tube or “pipette” is used to enter the shell and gently remove one of the blastomeres.  This blastomere will be handled in different ways depending on whether PGD will be done to determine chromosomal abnormalities (aneuploidy) or an inherited genetic disorder (single gene defects) in the embryo.

What are chromosomes and genes?  Chromosomes are structures found in the nucleus or center of a cell that contain all of our genes.  The genes are composed of DNA and are carried on the chromosomes.  Genes direct the production of all of the molecules that form the structures of a cell, and determine the inherited characteristics that distinguish one individual from another.

Human cells should have 23 chromosome pairs (46 total).  Two of the 46 are the sex chromosomes, which are the X and Y chromosomes.  Normally, females have two X chromosomes and males have one X and one Y chromosome.  During reproduction, each parent contributes 23 chromosomes.  The egg has 22 chromosomes plus an “X” chromosome.  The sperm has 22 chromosomes plus an “X” or “Y” chromosome, and determines the sex of the baby. 

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PGD for Aneuploidy

For chromosomal abnormalities (aneuploidy and translocations) the embryo biopsy procedure is followed by a technique called fixation, which adheres the blastomere’s nucleus onto a glass slide while at the same time removing all other cellular debris.  Later we attach “probes” to specific chromosomes contained in the nucleus using a method called fluorescent in situ hybridization (FISH).  A probe is a short sequence of single-stranded DNA that matches a portion of a gene on a chromosome.  A fluorescent dye is attached to the probe.  Specific groups of probes are made for each chromosome which labels that chromosome in its own unique color.  Since DNA is composed of two strands of complementary molecules that bind to each other like chemical magnets, the probe is able to bind to the complementary strand of DNA, wherever it may reside on a person's chromosomes.  This allows us to visualize specific chromosomes in the nucleus.   

If your embryos are aneuploid, they have too many or too few chromosomes.  This may cause them to stop developing at a very early stage, or they may continue to grow and implant, resulting in a biochemical pregnancy or miscarriage.  If however, chromosomes X, Y, 13, 18 or 21 are involved, the pregnancy may go to term resulting in the birth of a child with a condition such as trisomy 21 (3-chromosome 21’s) also known as Down syndrome.

 Aneuploid embryos do not look any different from “normal” embryos in terms of morphology or appearance.  Therefore, in order to determine whether the embryos have the correct or incorrect number of chromosomes, we must perform PGD.

What causes aneuploidy?

Aneuploidy is most often a result of incorrect division of chromosomes in the eggs of ageing women.  Females, at birth, have their entire allotment of eggs, and as they age, their eggs age as well.  When the eggs are finally recruited from a resting stage before ovulation, their chromosomes must undergo a certain number of divisions before fertilization occurs.  It is during these divisions that errors may occur, and can affect the development and viability of the fertilized egg or embryo.  

The risk of conceiving an abnormal baby increases with maternal age, from 1/385 at age 30, 1/179 at age 35, 1/63 at age 40 and at the age of 45 the chance to deliver an affected child is 1/19.  However, the frequency of aneuploidy in embryos is much higher than at delivery.  This difference in percentages of affected embryos versus live born is due to the fact that a pregnancy with aneuploidy is less likely to attach to the uterus or go to term.  Most will not implant or will be miscarried.  As such, the percentage of affected pregnancies is reduced over the course of the pregnancy due to the affected pregnancies that are lost.  The lack of implantation and loss rate of aneuploid embryos are believed to be the main reasons why pregnancy rates decrease with advancing maternal age.

Is PGD for aneuploidy appropriate for you?

• Are you 35 years or older?

• Apart from age, do you have a history of spontaneous abortions or abnormal pregnancies?

• Do you have a history of IVF failure?

• Do you require micro-surgical sperm retrieval for your treatment cycle? Patients with non-obstructive azoospermia have an increased rate of aneuploidy in their embryos.

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For couples having PGD for single gene disorders, sperm injection (ICSI) must be done to achieve fertilization in their IVF cycle so that sperm cells won’t accidentally be picked up during the embryo biopsy procedure.  The presence of sperm cells could alter the PGD result.  The embryo biopsy procedure is followed by the transfer of the cell or blastomere into a tube containing a solution that lyses the blastomere and removes all of the cellular material except the DNA.  Since individual genes contain relatively small amounts of DNA, the DNA from the single gene in question must be “amplified” or multiplied hundreds and hundreds of times in order to be detected.  Polymerase Chain Reaction (PCR) is a technique that amplifies the number of copies of specific regions of DNA in order to produce enough DNA to be sufficiently tested.  

What are some of the diseases that NFC can screen for using PGD technologies?

•  Achondroplasia (FGFR3)                  

• Adrenoleukodystrophy (ABCD1)

• Agammaglobulinemia-Brutona (TyrsKnse)

• Alpha-Antitrypsin (AAT)

• Alpha Thalassemia (HBA1)

• Alport Syndrome (COL4A5)

• Alzheimer (very early onset-PSEN1)

• Beta Thalassemia (HBB)

• Bloom Syndrome (Blm)                                    

• Canavan Disease (ASPA)

• Charcot Marie Tooth Neuropathy - 2E            

• Charcot-Marie-Tooth Neuropathy - 1B

• Choroideremia (CHM)                                       

• Chronic Granulomatous Dz (CYBB)

• Citrullinemia (ASS)                                            

• Cleidocranial Dysplasia (RUNX2)

• Congen. Adrenal Hyperplasia (CYP31A2)    

• Congen. Erythropoietic Porphyria (UROS)

• Crigler Najjar (UGT1A1)                                                    

• Cystic Fibrosis (CFTR)

• Darier Disease (ATP2A2) 

• Diamond Blackfan (DBA2)                                               

• Diamond Blackfan (DBA-RSP19)

• Duchenne muscular dystrophy (DMD)

• Dystrophy Myotonica (DMPK)                                           

• Emery-Dreifuss Muscular Dystrophy

• Epidermolytic Hyperkeratosis (KRT10)

• Factor 13 Deficiency (F13A1)

• Familial Adenomatous Polyposis (APC)       

• Familial Dysautonomia (IKBKAP)

• Fanconi Anemia A (FANCA)                             

• Fanconi Anemia C (FANCC)

• Fanconi Anemia F (FANC F)                            

• Fanconi Anemia G (FANCG)

• Fragile X (FMR1)                                                 

• Friedreich Ataxia I (FRDA)

• Gaucher Disease (GBA)                                   

• Glutaric Acidemia - 2A

• Hemophilia A (F8)                                              

• Hemophilia B (F9)

• HLA DRBeta1 Class II MHC (HLA DRB1*)                    

• HLA-A Class I MHC (HGNC HLA-A )

• Hunter syndrome (IDS)                                     

• Huntington Disease (HD)

• Hurler Syndrome (MPSI-IDUA)                                        

• Hyper IgM (CD40-ligand; TNFSF5)

• Hypophosphatasia (ALPL)                                               

• Incontinentia Pigmenti (KBKG-NEMO)

• Kennedy Disease (AR)                                                     

• Krabbe (GALC)

• Lesch-Nyhan (HPRT1)                                      

• Leukemia, Acute Lymphocytic (for HLA)

• Leukemia, Acute Myelogenous (for HLA)      

• Leukemia, Chronic Myelogenous (for HLA)

• Leukocyte Adhesion Deficiency (ITGB2)                        

• Li-Fraumeni Syndrome (TP53)

• Lymphoproliferative Disorder (X-linked)                        

• Marfan Syndrome (FBN1)

• Menkes (ATP7A)                                                 

• Metachromatic Leukodystrophy (ARSA)

• Mucolipidosis 2 (I-Cell)                     

• Neurofibromatosis (NF1 & NF2)

• Niemann-Pick type C (NPC1)                          

• Ornithine Transcarbamylase Deficiency (OTC)

• Osteogenis Imperfecta (COL1A1)                  

• Pachyonychia Congenita (KRT16 & KRT6A)

• Periventricular Heteropia (PH)                         

• Polycystic Kidney Disease (AR-PKD1)

• Polycystic Kidney Disease (PKD1)                 

• Retinoblastoma 1 (RB1)

• Rhesus blood group D (RHD)                         

• Rhizomelic Chondrodysplasia Puncta RCDP1

• Sacral Agenesis (HLXB9)                                 

• Sanfilippo A (MPSIIIA)

• SCID-X1 (SevereCmbndImmuneDefic (IL2RG)           

• Sexing for X-linked Dz (AMELX/Y; ZFX/Y)

• Shwachman-Diamond Syndrome (SBDS)                   

• Sickle Cell (HBB)

• Smith-Lemli-Opitz (SLOS)                                                

• Spinal muscular atrophy (SMN1)

• Spinocerebellar Ataxia2 (SCA2)

• Spinocerebellar Ataxia-3 (SCA3)     

• Tay-Sachs (HEXA)

• Treacher Collins (TOCF1)                

• Tuberous Sclerosis 1 (TSC1)Wiskott-Aldrich Syndrome (WAS)

***PGD has been performed for 130 different diseases - these are just a few***

If your disease is not listed, please call Amy Jones, MS (615-277-2448) for more information.

What are the risks of the PGD procedure?  

While PGD is a relatively new procedure offered to the IVF patient, the embryo biopsy technique itself employs methods (i.e. micromanipulation) that are already commonly used in an IVF lab.  The actual risk of damaging an embryo during a biopsy procedure is less than 1% in experienced PGD centers.  In addition, removal of one cell does not eliminate any part of the future fetus.  The embryo at this point is “totipotent” or has “all potential”.  This means that any single cell in an embryo up to approximately four days in culture has the ability to produce a baby.  In other words, the cells at this point are not differentiated.  With PGD, the chances of having a misdiagnosis are 10%.  However, a false negative result (the chance of classifying an abnormal embryo as normal) is only 3.5%.

What are the benefits of the PGD procedure?

By assessing your embryos for chromosomal abnormalities, we can avoid the risk of implanting an abnormal embryo into your uterus.  Aneuploidy screening avoids the transfer of embryos that would never implant due to chromosome abnormalities; thus maximizing your chances of getting pregnant in a single cycle.  Additionally, studies have shown that PGD for aneuploidy increases implantation rates, reduces the rate of pregnancy loss by half, and increases take-home baby rates.

How to have PGD

PGD is only performed in a very specialized IVF/Genetics centers.  Nashville Fertility Center has the latest technology for the entire procedure on-site, thus providing the patients with the best care available for these types of procedures.  If you are interested in PGD, please contact Amy Jones, MS director of Reproductive Genetics @ NFC (615-277-2448).

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References:

www.asrm.org/Patients/FactSheets/genetic_screening.pdf