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Preimplantation Genetic Diagnosis (PGD)
Overview articles
Quick Overview of Human Development
PGD Technical Description
Application to Fanconi Anemia
Odds of Success
Process Overview
Payment
Mutation Analysis
System Setup
IVF Cycle
Retrieval to Transfer
Day 0 - Retrieval, fertilization, potential
polar body biopsies for PGD
Day 1 - embryo development
Day 2 - embryo development
Day 3 - blastomere biopsy for PGD
Day 4 - genetic testing
Day 5 - test results & transfer
Pregnancy and Pregnancy Maintenance
PGD Providers
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Preimplantation Genetic Diagnosis: The Next Big Thing? by Ricki Lewis, The Scientist 14[22]:16, Nov. 13, 2000 A good overview. Unfortunately, they recently changed their policy and you have to pay to view archive content.
Preimplantation Genetic Diagnosis Fact Sheet. Has a nice diagram of the process.
Preimplantation Diagnosis for Fanconi Anemia Combined with HLA Matching, by Y. Verlinsky, S. Rechitsky, W. Schoolcraft, C. Strom, and A. Kuliev; JAMA, June 27, 2001, Vol. 285, No. 24, pages 3130-3133. Editorial on page 3143. An account (fairly technical) of the first time PGD was used to for both FA and HLA typing--the Nash case.
It will be helpful in to know a few terms related to human embryology. The egg
cell (an oocyte) prior to fertilization divides into two unequal cells. The
larger cell is a mature egg that will be fertilized. The small cell is called the first
polar body and contains some chromosomes from the egg. Upon penetration of the egg
with a 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 smaller cell is the second polar body. In the larger
cell, the sperm
and egg nuclei will fuse and the combination is now called a zygote.
The zygote then begins splitting (cleavage) into smaller and smaller blastomeres--each
cell being called a blastomere--increasing in cell count from 1, to 2, then 4, then 8
cells. Each blastomere becomes smaller and smaller with each subsequent
division. When it reaches 16 cells, it is called a morula. Cell division
continues, and after about 5 days a central cavity will be observed and the entire cell
mass is now called a blastocyst. The blastocyst will go on to implant in
the uterus; through subsequent growth, the name will change to embryo, fetus, and baby!
This site has good overview of human development.
Preimplantation Genetic Diagnosis (PGD) is a technique in which embryos obtained from in-vitro fertilization (IVF) are biopsied and tested for their genetic composition. Only those embryos that are free of genetic disease are transferred to the uterus to try and achieve a pregnancy.
PGD is possible because human development has the property of indeterminate cleavage. That is, if you remove a blastomere or two from an eight-cell human embryo, it will continue to develop into an embryo, then a fetus, and finally a baby. This one cell contains all of the genetic information of the embryo--so it can be tested for chromosome abnormalities or for specific disease causing genes.
Two technologies
have enabled PGD analysis. The first is fluorescence in situ
hybridization, or FISH. FISH uses DNA probes that have fluorescent labels
(fluorolabels) attached to them. The DNA probe joins together (hybridizes) with the
single-stranded DNA under study. When viewed under a microscope, the fluorescent
signals can be seen. Fluorolabels of different colors have been designed, making it
possible to hybridize a number of different probes to a single chromosome and distinguish
the individual signals.
The second technology is Polymerase
Chain Reaction (PCR) which is used to repeatedly copy (amplify) select regions of a
DNA molecule. For PCR, a pair of primers (short single-stranded DNA segments)
are needed that attach to either side of the target DNA to be copied. The target
DNA, the primers, a polymerase molecule which duplicates DNA, and a supply of nucleotides
are mixed together are go through a series of heating and cooling cycles. The segment of
the target DNA between the primers will be copied during each cycle, accumulating in the
mixture in an exponential fashion. After 30 cycles, the segment will have been
copied 250 million times. The results of PCR can then be analyzed in various ways, such as
agarose gel electrophoresis or sequencing.
These two techniques are used in PGD. FISH is very powerful for identifying chromosomal defects such as aneulploidy (extra or missing copies of single chromosomes). PCR is very powerful for detecting single-gene disorders. Your PGD provider will develop a system which combines a biopsy strategy (polar bodies, blastomeres), and an analysis strategy (using a combination of FISH and/or PCR) to achieve the analysis result you need.
PGD has been applied in two ways for couples who are carriers of pathogenic Fanconi Anemia genes.
First, you can clearly use PGD to have additional children that would not be affected by Fanconi Anemia. PGD is used to determine those embryos that have two copies of the pathogenic genes the parents carry--those embryos are affected by FA, so they are not transferred to the woman's uterus. Only the embryos that have only 1, or none, of the pathogenic genes are transferred. This is referred to "selecting against" Fanconi Anemia--you determine which embryos have FA, then choose the remainder.
Second, PGD can be used not only to select against FA, but also to select for an exact (6/6) HLA tissue match to an existing sibling that is affected by FA. The cord blood of the new baby could then be used for a bone marrow transplant (BMT) of the sibling with FA. This was first done by the Nash family. The genetic analysis done as part of PGD can also determine which HLA genes the embryo has, and thus, how close a tissue match is to the affected sibling. This is sort of the best of both worlds: a new baby not affected by FA, plus cord blood to transplant the affected sibling.
The Simple Answer
For two parents who are heterozygous for a pathogenic FA gene, recall that 1/4 of the offspring will be affected by FA, and 3/4 will be unaffected by FA. So if you use PGD to select against PGD, then approximately 3 of 4 embryos (or 75%) you test should be suitable for transfer into the uterus to achieve a pregnancy.
If you want to also select for a matching HLA type to a sibling, then you have to factor in the odds of an offspring being an exact HLA match. It turns out that it is 1/4 will be an HLA match, the remaining 3/4 will not be exact matches. So, the number of embryos that are not affected by FA and are an exact HLA match is the product of the two probabilities: 3/4 * 1/4 = 3/16. So you would expect 3 of every 16 embryos (or 18.75%) tested will be suitable for transfer to the uterus to achieve pregnancy.
The Complicated Answer
The sad truth is that you will not yield the number of embryos predicted from the simple Mendalian genetic calculations. PGD is done as part of an in-vitro fertilization (IVF) cycle, which has it's own success rates associated with it. There are several factors that will decrease the number of embryos that meet all of your criteria.
First, the fertilization rate. Not all of the eggs retrieved will successfully fertilize--only about 70% will fertilize using ICSI. Then, the zygotes have to make it to the blastocyst stage (day 5 of development). Using a state-of-the-art sequential culture media, the yield to the blastocyst stage is 50%. Let's compute (by multiplication) the yield with this factors considered:
Analysis Goal Mendal % Fertilization % Blastocyst % Overall Yield Fanconi only 75% 70% 50% 26.25% Fanconi + HLA 18.75% 70% 50% 6.5%
So what does this mean? If you're only using PGD to select against FA, then you should expect that 26% of mature eggs harvested will be available for transfer back to the uterus. If you're selecting against FA and selecting for a matching HLA type, then you should expect only 6.5% of the mature eggs will be available for transfer.
The process can also be very frustrating given the blastocyst yield. Consider: the biopsy is done on Day 3 (after fertilization), and the genetic testing is done during Day 4. You get the results on Day 5. A viable embryo that was doing well on Day 3, and meets your criteria, may not be alive on Day 5. So you get the results that you have an embryo meeting your criteria only to find out that it died while you were waiting for the test results.
Could it be much better than this? You might very well get a better fertilization rate, since you'll be using ICSI--say as high as 90%. However, you could get a much worse blastocyst rate, since the biopsies result in a lot of handling of the embryo--your blastocyst rate could be as low as 10-30%. The blastocyst rate is unlikely to be higher than 50% (some research has postulated that the maximum possible blastocyst rate for humans is 65%).
What does this mean? It means you need a lot of eggs if you want to do FA+HLA. The woman needs to be a good responder to the hyperstimulation drugs. Hopefully you could get something like 20 eggs per cycle. If you are only selecting against FA, then even a woman with low response (say, 10 eggs per cycle) to the hyperstimulation drugs could expect a blastocyst transfer every cycle.
A few more complicating factors. The pregnancy rate from blastocyst transfer is 50%. So if you get a blastocyst meeting all of your criteria, you have only a 50% chance of achieving a pregnancy with it. If you do achieve a pregnancy, your risk of miscarriage is about 30% (higher if you are older). Although the fact that you had performed PGD may help reduce the miscarriage rate somewhat, as many miscarriages are due to genetic abnormalities in the embryo. Here is a table that puts all the factors together to come up with the expected live birth yield (analysis yield is from the last column in the table above):
Analysis Goal Analysis Yield % Pregnancy Yield % Miscarriage Yield % Live Birth % Fanconi only 26.25% 50% 70% 9.2% Fanconi + HLA 6.5% 50% 70% 2.3% Recall that you need to multiply this by the number of mature eggs harvested. So if you have 20 eggs, then your expected number of live births by selecting only against Fanconi Anemia is 20 * 0.092 = 1.84 babies.
Note that this is the expected live birth yield for the entire process; if you get through part of the process with better results, it carries forward. For example, if you have 2 blastocysts to transfer, then your odds of getting pregnant with at least one child is 75%, then factoring in the miscarriage yield of 70% (i.e., 30% miscarriage rate), your odds of having a live birth are 52.5%. If you get further down the process--you get pregnant with 1 child--then your odds of having a live birth are 70% (the miscarriage yield).
So are the odds of a Fanconi + HLA match really so low?
Consider this study, which used PGD to select against another genetic disease, ß-thalassaemia, and for an HLA match. Two families went through six cycles, generating 58 embryos for testing (or, an average of about 10 per cycle). The analysis technique was successful on 54 of the 58 (testing yield of 54/58 = 93%, which isn't even factored in the tables above). Two transfers were done and one pregnancy was obtained (note: they didn't say birth). So how did they do? Out of 58 embryos, two, or 3.4%, were able to be transferred. As for pregnancies, only 1 of the the 58, or 1.7%, resulted in a pregnancy.
There is also this study which used PGD to select against ß-thalassaemia (14 cases), Wiscott-Aldrich syndrome (1 case), and leukemia (2 cases). The families went through 17 cycles, generating 266 blastomeres (an average of 15.6 per cycle). Of those, the analysis technique was successful in 255 of those 266 (testing yield of 255/266 = 95.9%). Out of those, 14 (5.2%, 14/266) were both HLA matches and unaffected by genetic disease. These 14 were transferred into patients, resulting in 4 pregnancies (1.5%, 4/266). Three (1.1%, 3/266) of those pregnancies (one twin, two singletons) were continuing at the time the article was written (note this means 1 of the 4 women had a miscarriage, 1/4 = 25% miscarriage rate).
Sorry to be so glum on the odds of success here, but you should know that PGD is at the cutting edge of reproductive technology. Being a cutting edge technology, the expense is high and the success rate isn't so great. Be prepared to go through multiple cycles.
This section will cover all of the steps, in order, that you need to go through in pursuing IVF+PGD.
Your first step should be to contact a PGD provider and starting working with them. Your PGD provider can help guide you through many of the steps below.
The first step is to take a dumptruck full of cash and dump it on the the front lawns of several doctors. I'm not kidding.
First, there are some one time charges at the beginning for genetic analysis and many tests for IVF. The mutation analysis may be about $4,000. The IVF-related tests may run $1500. Getting your PGD system setup (which includes some genetic analysis) will be about $4,000. So that's about $10,000 in up-front costs.
IVF cycles with PGD are not cheap. IVF may cost you $15,000 per cycle, plus $3,000 for drugs. The PGD will be $2,500 for a single gene (FA), and $5,000 for two genes (FA + HLA), plus up to $2,000 in travel charges for the embryologist who will do the biopsies. If you need to travel for the IVF cycle, you have to add in travel expenses (say, $3,000).
So it could cost you $25,000 per cycle to attempt IVF+PGD. All providers will want cash upfront before providing services. And there are no guarantees.
For PGD, you will need to know the exact nature of the mutations affecting your child with FA--that is, the exact nature of the pathogenic mutations that you, as parents, are carriers for. Remember that it is unlikely that you and your partner have exactly the same mutation--you most likely each have a different kind of pathogenic mutation. This information on the mutation is used to setup a genetic analysis system for the biopsies done to your embryos (see system setup, below).
Follow the link above for more information on the mutation analysis process. You should expect the mutation analysis to take 10-12 weeks (a lot less if your family is afflicted by FANCC mutations).
If you are going to be testing for an HLA match as part of your PGD, then your PGD provider will need to perform an analysis technique called "linkage analysis" on your family to determine how the HLA inheritance works in your family. A prerequisite to this is to have high resolution HLA typing done on the mother, father, and affected child. This HLA typing information, along with a DNA sample from each person, will allow your lab to perform linkage analysis. Linkage analysis basically determines which other genes are inherited with high probability with your HLA genes. This information is used to predict the HLA type of the embryos being tested. You should expect the linkage analysis take to 2-3 months.
Once you have your mutation analysis done, and the linkage analysis done (if you are also testing for HLA), your PGD provider will need some time to "setup the system." This means they need to take the analysis data and figure out an experimental method used to test the genetic material biopsied from your embryos. This should take 2-3 months, perhaps less.
So from the start of your desire to pursue PGD to this point (analysis system is setup), it's taken you 6-9 months. Don't be discouraged if it takes an entire year.
PGD is done as part of an in-vitro fertilization (IVF) cycle. Prior to your first cycle, your IVF provider will require several tests to be performed. After these tests are done once, they are typically not needed for subsequent cycles within the next year.
The woman will typically begin the IVF cycle by going on birth control pills! This is done to regulate the woman's cycle. After being on birth control pills for one month, they are stopped. The woman then has a period.
"Day 1" is the first day of the period.
On Day 21, the woman will start injecting lupron. Lupron is a medication that prevents premature ovulation by suppressing pituitary secretion of LH and FSH. LH is Luteinizing Hormone, a hormone produced by the pituitary gland that triggers ovulation. FSH is Follicle Stimulating Hormone, another hormone produced by the pituitary that stimulates the ovary to ripen a follicle for ovulation.
After another 10-14 days, the woman gets another period.
A baseline ultrasound is then scheduled. A couple of days after the ultrasound, stimulation will start.
Stimulation is done by lowering the dose of lupron and injecting other drugs (fertinex, follistum, gonal-F, repronex, etc.) that stimulate the ovaries to produce eggs. The woman will be on these medications for 10-12 days. While undergoing stimulation, the woman will be monitored by blood tests (to check estrogen levels) and ultrasounds (to check the number and size of follicles).
When the follicles reach a size of 18-20 mm (measured by ultrasound), you will be given instructions to take the hCG shot (the "trigger shot") and you will stop taking lupron and other medications. hCG is Human Chorionic Gonadotropin, which prepares eggs for ovulation.
2 days later will be egg retrieval.
Note that the above stimulation protocol is only an example. The actual protocol will vary from patient to patient. Your doctor will develop a specific schedule just for you that instructs you on exactly which medications to take when, when to come in for tests, etc.
Two days after the hCG shot (the "trigger shot"), the woman will undergo egg retrieval. The eggs will then be fertilized using ICSI, the cultured through the first 5 days of development. Key events are listed by day, below:
Day 0 - Retrieval, Fertilization, potential polar body biopsies for PGD
The day of the egg retrieval is Day 0. On Day 0, the eggs will be retrieved from the woman by aspirating each follicle with a needle under the guidance of ultrasound. The woman is sedated during this procedure.
The retrieved eggs are cultured for 6-20 hours depending on egg maturity.
The eggs will divide into two unequal cells. The larger cell is the mature egg that will be fertilized. The smaller cell, called the first polar body. This polar body may be removed by the embryologist for genetic analysis, depending on the requirements of your PGD system.
On this day, the man also gets to do something. He needs to provide a sperm sample.
Once the eggs are mature, the embryologist will inject a single sperm into each mature egg by a procedure called intracytoplasmic sperm injection (ICSI). This procedure basically involves pulling a single sperm into a microscopic hollow needle, poking this needle into the egg, then injecting the sperm into the egg. You should expect about 70% of your eggs to be successfully fertilized by this method.
After fertilization, the egg undergoes another unequal cell division. The larger cell will join with the sperm's genetic material to create a pre-embryo (the zygote). The small cell is the second polar body. This second polar body may also be removed by the embryologist for genetic analysis, again depending on the requirements of your PGD system.
Day 1 - Embryo development
The day after egg retrieval and fertilization is Day 1. On this day, the embryologist monitors the embryos as they undergo cleavage as part of their development.
Day 2 - Embryo development
The embryo continues to undergo cleavage.
Day 3 - blastomere biopsy for PGD
On Day 3, your embryos should have reached the 8 cell stage--8 blastomeres. If your PGD system requires it, one blastomere (cell) will be removed by the embryologist for genetic analysis.
Day 4 -- genetic testing
Your embryos continue to develop by undergoing cell division, steadily increasing the cell count.
Your PGD provider is also busy this day doing the genetic testing of the biopsied materials. The genetic testing of the polar bodies may have begun earlier (days 1 & 2), but they only have Day 4 for analyzing the genetic material of the blastomeres that were biopsied on Day 3.
Day 5 - test results & transfer
On this day, you will find out:
- The genetic test results of all the embryos biopsied. You will know which ones are affected by FA, which ones are carriers of 1 pathogenic FA gene, and which ones are carry no pathogenic FA genes. Ones suitable for transfer are the carriers and the non-carriers. If you also tested for HLA, you will also find out which ones are a perfect HLA tissue match to your affected child.
- You will also find out how many of your embryos survived from Day 3 until now. You should expect at least 50% of the embryos will have died by the time you reach Day 5.
This can be a heartbreaking day. You may find out that you had an embryo that is unaffected by FA and is an exact tissue match for your affected child, but that this embryo did not survive to Day 5! Remember the odds of success.
If you are lucky, and have probably undergone a lot of IVF+PGD cycles, you may have an embryo that survived to Day 5 that you want to try and achieve pregnancy with. This embryo (or embryos if you're lucky) will be transferred into the woman's uterus via a catheter. The woman will then have to say immobile for about 30 minutes. The woman can then go home to bed rest for the next 24-48 hours (follow your doctor's requirements).
You may also on this day choose to freeze some embryos for transfers at a future date.
Ten to twelve days after the embryo transfer, you will have a pregnancy test. This will be a blood test. If it is positive, you should expect it to be repeated two days later. The second pregnancy test helps to rule out a chemical pregnancy. The pregnancy test is measuring your hCG level in your bloodstream; in a true pregnancy, this level should double every two days.
Pregnancy Maintenance: You will have been taking progesterone injections, or a cream, or suppositories from the day of your hCG shot (trigger shot) until the day of your pregnancy test. If the pregnancy test is positive, you will need to continue the progesterone for another 6-12 weeks. You may also need to take estrogen for several weeks. You will go in for periodic blood tests to monitor the levels of estrogen and progesterone in your blood; adjustments in the doses of these drugs will be made based on the blood test results.
Other PGD providers other than those listed may be able to do it for Fanconi Anemia as well.
Reproductive
Genetics Institute
2825 N Halsted St
Chicago, IL 60657
Tel. (773) 472-4900
FAX (773) 871-5221
email: 
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Last updated: 31 Aug 2005
