Embryo Testing in IVF

This is information about embryo testing that is available in the IVF process.  This is from The Advanced Fertility Center of Chicago http://www.advancedfertility.com/preimplantation_genetic_diagnosis.htm

PGD and IVF - Preimplantation Genetic Diagnosis & In Vitro Fertilization, Pros and Cons About PGD and PGS

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What is PGD, or preimplantation genetic testing?

PGD, preimplantation genetic diagnosis, is the process of removing a cell from an in vitro fertilization embryo for genetic testing before transferring the embryo to the uterus.
The term PGD, preimplantation genetic diagnosis, is often loosely used to refer to any testing performed on an embryo prior to it being transferred to the uterus. However, the distinction should be made between the terms PGD and PGS.
  • PGD, preimplantation genetic diagnosis, involves removing a cell from an IVF embryo to test it for a specific genetic condition (cystic fibrosis, for example) before transferring the embryo to the uterus.
  • PGS, preimplantation genetic screening, is the proper term for testing for overall chromosomal normalcy in embryos. PGS is not looking for a specific disease diagnosis - it is screening the embryo for normal chromosome numbers.
History of PGD - PGS
IVF, in vitro fertilization, was first successfully used in 1978. It was not until years later that scientists began tinkering with the possibility for extracting one or more cells from the embryo to get information about the potential health of the child that might result following implantation of that embryo.

The first report of pre-implantation genetic testing in humans with a pregnancy resulting was published in 1990. Major improvements in these technologies have been developed since then. Both the embryo biopsy techniques as well as the genetics technologies used on cells removed from embryos have improved dramatically.
  • Much has been learned in the PGD/PGS field over the past 20 years - and much remains to be learned
Different stages of egg and embryo development can be biopsied
There are 3 basic stages at which eggs or embryos are biopsied at the present time.
  1. Polar body biopsy is performed at an early stage when the polar body of the egg is removed and can have its genetic material tested. A mature egg has one polar body and a fertilized egg has 2 polar bodies. Either the first polar body of the egg or the first and second polar bodies can removed for testing.
  • This technique does not involve involved taking anything from the embryo from the cells that would become part of the fetus or the placenta. However there is still potential for damaging the developmental potential of the resulting embryo with polar body biopsy.
  • There is some evidence that the polar bodies may help direct differentiation of the cells in the very early embryo. They may be involved in determination of which cells become precursors to the fetus (inner cell mass) and which cells become present precursors to the placenta (trophectoderm).
  1. Biopsy at the cleavage stage is done on day 3 of embryo development. At this point the embryo usually has 6 to 10 cells.
  • Removal of a portion of the zona pellucida (outer shell of embryo) is performed
  • Then 1 or 2 cells is pulled out of the embryo for subsequent testing
  • The day 3 cleavage stage biopsy technique has been shown in several studies to be detrimental to embryo development. Therefore, many IVF programs are moving away from it in recent years.
  1. The third type of PGD biopsy is called trophectoderm biopsy. It is performed at the blastocyst stage after the embryo has differentiated into an inner cell mass, a trophectoderm component and a fluid filled cavity.
  • With trophectoderm biopsy at the blastocyst stage a small hole is made in the shell of the embryo and several cells that are precursors to the placenta (trophectoderm) are removed for testing
  • This technique has shown promising results in some US IVF programs. It is now considered by many experts to be the biopsy procedure of choice for PGD and PGS testing.

PGD, Preimplantation Genetic Diagnosis for Genetic Disorders
Preventing genetic diseases by testing IVF embryos

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PGD for single gene defects to prevent transmission of genetic disease

Pre-implantation genetic diagnosis is a technique that is used along with in vitro fertilization, IVF and allows testing of embryos for certain characteristics such as their chromosomal makeup and also testing for genetic diseases that are passed on through families.

When one or both partners in a couple are carriers of the genetic mutation that could leave could lead to a serious medical condition in the child in vitro fertilization and preimplantation genetic diagnosis testing can be performed on their embryos.

In past years couples that carried a genetic mutation could choose between not having children or having prenatal testing done with either amniocentesis at about 16 to 18 weeks of pregnancy or with chorionic villus sampling at about 11 to 12 weeks to see if the baby was affected with the genetic condition. The couple then has the option of terminating the pregnancy if the baby is affected with the disorder.

Now couples that are carriers for genetic diseases have the option of having IVF and PGD to screen their embryos prior to transferring them to the uterus.

Not all diseases can be tested for in this manner. Single gene disorders are caused through the inheritance of a defective gene. These disorders are classified as either recessive or dominant. A recessive disorder requires 2 bad copies of the gene to pass the disease on to the baby. With a dominant single gene disorder only one copy of the defective gene is needed to cause the disease.

There are over 1000 single gene disorders that have been identified at the present time. Many of these disorders are very rare. However, some are so common in certain ethnic groups that routine screening to check whether someone is a carrier is recommended prior to getting pregnant. This is often referred to as carrier genetic testing (or screening).

The most common single gene disorders that PGD has been used for are:
  • Cystic fibrosis
  • Tay-Sachs disease
  • Spinal muscular atrophy (SMA)
  • Hemophilia
  • Sickle cell disease
  • Duchennes muscular dystrophy
  • Thalassemia
However, there are hundreds more genetic diseases that can have single gene testing done using IVF and PGD.

A partial list of relatively common single gene diseases is below.
Autosomal recessive disorders
Sanhoff disease, Gaucher disease, adenosine Deaminase deficiency, glycogen storage disease, Fanconi anemia, adrenal hyperplasia, phenylketonuria (PKU).
Autosomal dominant disorders
Neurofibromatosis, Von-Hippel Lindau, myotonis dystrophy, Huntington's Disease, Marfan syndrome, osteogenesis imperfecta, Charcot-Marie-Tooth, APP early onset Alzheimers, polycystic kidney disease, retinitis pigmentosa, familial adenomatous polyposis, achondroplasia.
X-linked disorders
Ornithine carbamyl transferase deficiency, Fragile X, X-linked hydrocephalus.

How is IVF and PGD with genetic testing done?
In vitro fertilization is performed, the embryos have trophectoderm biopsies done on day 5 and 6 at the blastocyst stage of development.
The biopsied cells are then sent to the genetics laboratory for evaluation. The genetics laboratory does their fancy genetics magic and determines whether the embryo contains the "abnormal" DNA of the genetic disease in question, or not.
The testing is done utilizing high tech genetics technology that analyzes the DNA sequence in the cells for the specific gene in question.

PGD genetic testing for cystic fibrosis
An example of using PGD for genetic testing for single gene defects is the situation where a couple are both carriers for the recessive gene that causes cystic fibrosis. In order for a child to have cystic fibrosis it needs to get one copy of the abnormal recessive gene from each parent.
A normal gene combined with an abnormal gene does not cause the disease. That person would only be a carrier and would not affected by cystic fibrosis. If both parents are carriers of this recessive genetic condition then every child they have has a one in 4 risk for having the cystic fibrosis disease.
Therefore, sometimes a couple that are both known carriers for the abnormal cystic fibrosis gene will decide to undergo in vitro fertilization with PGD genetic testing of their embryos for cystic fibrosis.
  • In such a case we expect that one out of 4 embryos would have both of genes being abnormal which would lead to a child with the cystic fibrosis disease
    • Those abnormal embryos would not be transferred back to the mother's uterus
  • We expect 2 out of 4 embryos to be carriers for the abnormal gene (one normal copy and one abnormal)
  • We expect 1 out of 4 to have 2 copies of the normal gene (not carriers)
  • Embryos would be chosen for transfer to the uterus that genetic testing showed did not have 2 copies of the abnormal cystic fibrosis gene.
Blastomere biopsy on day 3 after fertilization is one way that PGD testing can be done
With blastomere biopsy, a slit is made in the shell of the embryo on day 3 and a pipette is introduced into the embryo. Then one of the cells is carefully teased out of the embryo which would have approximately 8 cells at that time.

If it's an 8 cell embryo and we remove one cell for testing, it then is a 7 cell embryo. At this stage of development, all of the cells are undifferentiated. Therefore, losing one cell would not take anything away from the subsequent child. The cells are all clones of each other at this point and would continue dividing and eventually differentiate into specific cell types.

The biggest problem with day 3 blastomere biopsy is that it has been shown to result in a significantly reduced implantation potential after the embryo is transferred to the uterus.

Trophectoderm biopsy on day 5 or 6 at the expanded blastocyst stage is a better way to do embryo biopsy
This is a newer technique that utilizes biopsy at a later stage of embryonic development. At the blastocyst stage there has been some differentiation of the cells into an inner cell mass (destined to become the fetus) and the trophectoderm (precursor to the placenta). A small portion of the trophectoderm cells are removed for the biopsy. The inner cell mass is not disturbed.

A disadvantage of trophectoderm biopsy is that with current technology it takes about 24 hours to get results back from the biopsy. Therefore, since the embryos should be transferred by day 5 or early on day 6 it is not currently practical to do trophectoderm biopsy and transfer fresh embryos to the uterus.
Therefore, trophectoderm biopsy is performed and the embryos are frozen and subsequently thawed and transferred later after results of testing are available.

Trophectoderm biopsy is less traumatic to the embryos than blastomere biopsy and therefore results in a higher success rate.

Preimplantation genetic screening - PGS for aneuploidy with IVF

Does PGS with chromosomal tests of embryos improve IVF success?

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What is PGS, preimplantation genetic screening?

  • PGS, preimplantation genetic screening, refers to removing one or more cells from an in vitro fertilization embryo to test for chromosomal normalcy
  • PGS screens the embryo for normal chromosome number
  • We can think of PGS as chromosomal disorder screening on IVF embryos
  • Humans have 23 pairs of chromosomes - for a total of 46
  • Down syndrome has an extra chromosome 21, referred to as trisomy 21. This should be detected by PGS.
  • PGS does not test for a specific disease - such as for cystic fibrosis - that process is referred to as PGD (preimplantation genetic diagnosis)
Many human embryos are chromosomally abnormal
Several studies have shown that overall about 50% of human preimplantation embryos from IVF are chromosomally abnormal. The rate of abnormalities is affected greatly by female age, as shown in the graph below. Chromosomes in eggs from older women have a significantly increased rate of abnormalities.

Rate of chromosomally abnormal human eggs and embryos by age

To a great extent, chromosomal abnormalities are responsible for failure of implantation of IVF embryos. Chromosomal abnormalities are also responsible for about 70% of miscarriages in early pregnancy.

What to do if the first IVF attempt fails
Problems in the past with aneuploidy screening of IVF embryos
IVF and PGS for aneuploidy (an abnormal number of chromosomes) has been used at some clinics in the United States since the mid 1990s. However, studies showed that performing embryo biopsy on day 3 and performing the genetic analysis using FISH technology (fluorescent in situ hybridization) did not result in an increase in the chance for the patient to have a successful IVF cycle.
There were 2 main problems with that approach.
  1. FISH technology was usually looking at only 5 chromosomes out of 23. Therefore, the FISH test would miss many chromosomal abnormalities. This resulted in abnormal embryos being transferred after the screening test came back "normal".
  2. The biopsies on day 3 were removing a cell (or 2 cells) from a 6 to 10 cell embryo. This required a relatively large hole being made in the shell of the embryo and then removal of a significant percentage of the "biomass" of that embryo (one sixth to one tenth of it removed, or more).
Recent advances allow for better IVF success rates after aneuploidy screening
Improvements in genetics technologies
Advances in the field have led to utilization of improved genetics technologies that allow assessment of all 23 pairs of chromosomes.
There are currently 3 technologies that can be utilized for assessment of normality of all 23 chromosomes:
  1. Array Comparative Genomic Hybridization (aCGH)
  2. Single nucleotide polymorphism microarrays (SNP)
  3. Quantitative real time polymerase chain reaction(qPCR)
Comparative Genomic Hybridization (often referred to as CGH) is a microarray technology that is often used now instead of the older and less comprehensive FISH. With microarray CGH, the actual DNA in the embryo is compared to a known normal DNA specimen utilizing thousands of specific genetic markers. This gives a more accurate result, with far fewer false normal or false abnormal results.

Some studies have determined that the error rate using array CGH technology is about 2%. FISH has an error rate of about 5-10%. Additionally, many other abnormal embryos would be reported by FISH as normal because the abnormality was in a chromosome that was not part of the FISH panel being used.

Improvements in embryo biopsy techniques
Trophectoderm biopsy is done at the blastocyst stage on day 5 and 6. At this stage there are many more cells present in the embryo. This allows multiple cells to be removed from the trophectoderm (precursors to the placenta). The inner mass cells (precursors to the fetus) can be left undisturbed during the biopsy.

With trophectoderm biopsy, about 5 cells are snipped off for testing. This does not significantly weaken the embryo because it has about 100 cells at this stage.

The combination of these two modifications (advanced genetics and trophectoderm biopsy) has led to significantly improved pregnancy success rates for patients that want to utilize PGS for their IVF treatment.

Some clinics in the US have been using trophectoderm biopsy and the newer genetics technologies to screen embryos in some IVF patients. The results seen in some IVF programs (including ours) have been quite promising.
  • We are seeing substantially improved ongoing pregnancy rates in patients that are having trophectoderm biopsy performed at the blastocyst stage with subsequent freezing of their embryos.
  • A frozen thawed transfer cycle is done after the chromosomal analysis results come back
Uterine receptivity issues
There is some interesting speculation that the uterine lining could be less receptive during a stimulated cycle as compared to the controlled or "artificial" embryo replacement cycle.
  • Some fertility doctors believe that transferring embryos in a controlled cycle (using frozen embryos) gives a higher pregnancy rate than in a "fresh" cycle
  • This has not yet been carefully studied with multiple controlled clinical trials (as of 2014)
  • The improved success rate seen following blastocyst biopsy and comprehensive chromosomal analysis is mainly due to the benefit of transferring chromosomally normal embryos
  • There is probably some additional benefit derived from transferring the embryos in a frozen thawed cycle rather than in a stimulated cycle
Which couples should we be offering aneuploidy screening to?
  • This is currently an evolving issue in the field of reproductive medicine
  • Some potential candidates could be:
    • Women over (about) 35 years old that want IVF and make enough embryos so that they will have multiple blastocysts available for biopsy.
    • Patients at any age that have failed multiple IVF cycles. They want answers about why they are failing. They also want to know what to do in order to improve their chances to have a baby.
    • PGS can sometimes provide answers in these cases.
    • PGS can also can provide a "weeding out" of the abnormal embryos. For example, if one embryo out of 6 is chromosomally normal and 5 out of 6 are abnormal - we transfer the one normal embryo and should have a very good chance for a baby.
    • Couples with recurrent unexplained miscarriages


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