You may have heard about Preimplantation Genetic Screening as a technique provided in the IVF laboratory, and may have wondered if this technology is one you should consider incorporating in your IVF cycle. When considering various technologies in your IVF cycle, it is always important to clearly define what information you wish to gather with this technology, and also understand the pitfalls of the technology.

We have two methods of screening embryos. The first is called Preimplantation Genetic Diagnosis, for couples that have a known and defined genetic disease (e.g. Cystic fibrosis, Huntington’s disease, thalassemia), or are carriers of a single chromosome abnormality (chromosomal translocation). In this case we screen the embryo(s) for that particular genetic disorder, and transfer appropriate embryos. For this type of genetic screening, the aim is to conceive a healthy, unaffected child.

The other type of genetic screening is called Preimplantation Genetic Screening, where we screen the embryos for abnormalities in chromosome number. We all have 23 pairs of chromosomes. Embryos that have extra or missing chromosome(s) (aneuploid embryos) are much more likely to not implant, or to produce a miscarriage. The incidence of implantation failure, or of miscarriage, depends on which chromosome(s) are missing or duplicated. We therefore can screen an embryo with a “five or nine chromosome panel.” At PFC we utilize the nine chromosome panel. We look at the nine chromosomes that have been identified as most commonly being associated with implantation failures or miscarriages to see if that particular embryo has the correct number of those nine chromosomes. If so, this embryo is deemed “normal”, and can be transferred back to the uterus.

So who might consider PGS? Patients who have had a number of failed IVF cycles (documented failed implantations), those with a poor response to ovarian stimulation or those with poor embryo development (poor responders), those with recurrent miscarriages (>2 first-trimester miscarriages), those with a prior aneuploid pregnancy, those who are at least 35 years old are all candidates for PGS. The chances of improved pregnancy rates with PGS are dependent on the indication for PGS.

When we started doing PGS for various indications, we expected a dramatic improvement in implantation rates, and therefore pregnancy rates, as we were transferring pre-selected embryos. As it turns out, we have not necessarily seen those expected improvements in all patient groups. Patients who are younger than 35 yeas of age have a better chance at improved implantation and pregnancy rates using PGS. Improvements can still be obtained for older patients, if the 9 chromosome probe set is used (some centers use a 5 chromosome panel). Studies now indicate that patients who have at least 6 fertilized eggs to screen will also have a better prognosis than those with 5 or fewer. For those patients who have five or fewer fertilized eggs in their IVF cycle, we may actually recommend not proceeding with the PGS. In this case less manipulation of embryos may provide the patient with the best overall chance at pregnancy. Patients who have had less than 3 failed IVF cycles may have greater benefit from PGS than those with > 3 failed cycles. Patients with a prior aneuploid pregnancy or with recurrent pregnancy losses can also expect an improvement.

For patients who have had repetitive IVF cycle failures, or repetitive pregnancy losses, a PGS cycle may be diagnostic (explain if those failures/losses are from a high number of abnormal embryos), and in that sense may provide important information that explains those fertility failures. With those answers, the patient can then decide about pursuing similar treatment cycles, or choosing other options (using a donor egg, pursuing adoption, or choosing to live child-free). Studies indicate that results from one PGS cycle are indeed predictive of probable results in subsequent PGS cycles. In other words, if we have a cycle with a higher than expected percentage of abnormal embryos, we have to anticipate that we will probably have a similar result in subsequent PGS cycles.

There are many proposed reasons to explain why we are not achieving a higher implantation/ pregnancy rate in PGS cycles. There clearly is added stress placed on the embryo(s) when one cell is biopsied out, and when the embryo is kept in culture for an extra day or two while waiting for the results of the genetic testing. We currently can only test for 9 chromosomes, and it is possible that there may be undiagnosed abnormalities on one of the untested chromosome pairs. There is also a small possibility that an embryo we deem “normal” may actually not be normal (false negative result). It also may be that simply looking at chromosomes is not the final answer. Most likely the integrity and health of the cytoplasmic structures, and other important structures of the egg are also critical in the ability of the embryo to develop into a viable and healthy pregnancy.

Who Benefits Most?

• Patients with < 3 failed cycles, and > 5 fertilized eggs
• Patients 35 year and older (if using a 9 chromosome panel)
• Patients with a history of recurrent pregnancy losses
• Patients with a previous aneuploid pregnancy
• Patients using PGS as a Diagnostic Tool for:
  - Repeated IVF failure
  - Non-obstructive Azospermia

So, while PGS is a wonderful tool that can be incorporated into the various techniques of your IVF cycle, you need to be aware of the strengths and limitations of PGS testing. Your physician can help guide you in terms of the appropriate use of PGS and whether you may benefit from incorporating PGS in your IVF cycle.

A recent development in the laboratory at PFC is the acquisition of a laser for use in key procedures. The laser will be used to assist in the processes of Assisted Hatching (AH), Intracytoplasmic Sperm Injection (ICSI), and Pre-implantation Genetic Diagnosis/Screening (PGD/PGS).

All of these procedures require us to make a small opening in the outside shell of the egg called the Zona Pellucida (zona). Prior to laser technology this opening was made with an Acidified Solution, which would slowly dissolve away part of the zona until a small opening was achieved. Now with the laser, a beam of light creates a precise opening in the zona.

Laser use for PGD: The red “pilot light” marks target for the laser. The white circle marks a “safe zone”. The laser is usually fired 3 times for assisted hatching and 5 times for PGD embryo biopsy.

The zona pellucida is a non-living, but important part of the egg. It specifically allows only 1 sperm through to fertilize the egg, and then immediately hardens, preventing other sperm from getting in. After fertilization, the egg divides into 2 cells, and then these divide again into 4 cells. As the embryo continues through these rounds of cell division, the zona keeps all the cells together, since it encloses the embryo. After 5 or 6 days, the embryo has enough cells to begin forming a placenta and the embryo hatches from the zona and attempts to implant in the uterine lining.

Assisted hatching (AH) is a procedure that has been around for about 15 years and it is something that is often performed in the laboratory just prior to an embryo transfer procedure. It is a simple and precautionary procedure where we create a small hole in the zona just before transferring embryos to the uterus. Since the zona is not a living part of the embryo, making a hole does no harm, and in fact facilitates the embryo in hatching from the zona once it’s in the uterus. A normal embryo should be able to hatch all by itself, but in some patients we perform this procedure just to make sure a problem doesn’t arise when the embryo tries to escape from its shell. For AH, the laser will allow us to refine the procedure considerably. Firstly, we will be able to make a hole of an exact size, and secondly, the procedure will be performed more quickly and we will therefore further reduce the amount of time that an embryo is being handled. Traditionally, AH takes about 5 minutes per embryo, but with the laser this time will be reduced to less than a minute. For the process of embryo biopsy for PGD/PGS, an extremely precise opening is made in the zona to facilitate the removal of one cell. Again, the laser will speed the procedure up considerably and reduce the time that we’re working on each embryo.

By now you might be wondering if there are any harmful effects of using laser light on embryos. According to several studies and expert opinions, laser-assisted hatching is superior to chemical-assisted hatching as seen by improved development of “hatched” embryos to the blastocyst stage (the stage at which an embryo will implant in the lining of the uterus). Furthermore, laser-assisted biopsy of cells from embryos for PGD analysis does not appear to have a detrimental effect on the continued development of the embryos versus embryos not undergoing any biopsy procedures. This indicates that using a laser to do the biopsy procedure appears to be safe.

Current lasers have several built-in safety features. The laser system is equipped with a second non-laser beam of light, similar to a penlight, which allows the embryologist to observe where an opening of the zona would be created prior to firing the laser. Also, the temperature that the embryo is exposed to is controlled by the use of Isotherm rings. Isotherm rings help the embryologist prevent potential harmful thermal effects on cells adjacent to the zona due to heat from the laser beam. The rings indicate both the drill hole size and the safety region based on temperature. With this interactive feature, the user can predetermine the hole size and eliminate practically all risk of impacting cells within the embryo.

PFC’s new laser system has been tested for both accuracy and precision. In addition, the lab staff is undergoing training with Laser professionals on its use and maintenance. They will have unlimited practice time, ensuring the highest level of safety and technique when it comes time to use it on human embryos.

– Jean M. Popwell, PhD TS (AB, PFC Lab Embryologist)

We are the parents of a little boy with a rare, life-threatening, enzymatic disorder. He inherited this condition through genetic mutations passed along to him by us. Caused by a recessive genetic defect, neither of us is affected in anyway as we are simply “carriers” of this disease. Once we were determined to be carriers, however, we learned that should we conceive another child naturally, there is a 25-percent chance that we will have another affected child.

It had always been our plan to have more than one child. However, as we began to discuss the possibility of having a second baby, we both realized that given the physical, emotional and financial costs of being affected by this disorder, we were not comfortable with consciously bringing another child into the world without doing everything we possibly could do to avoid this for any other child.

Embryo Biopsy

After two years of extensive pre-conception counseling, we decided in-vitro fertilization (IVF) with pre-implantation genetic diagnosis (PGD) through Pacific Fertility Center would give us the best possible chance of giving birth to an unaffected child.

A medical technique whereby embryos can be screened for specific genetic defects prior to transfer to the womb, PGD has been performed for over 10 years and has proven to be a most effective method of diagnosing embryos for known genetic mutations. To-date there have been over 2500 PGDs performed around the world resulting in over 1600 children born without the disease for which they were screened. The error rate for PGD is less than two-percent; therefore, PGD would reduce our chance of having an affected child from 25% to less than 2%.

A little over a year ago we began our IVF with PGD Embryo Biopsy cycle. On Day 3 after retrieval, when our embryos were eight-cells or so in size, a single cell was biopsied from each embryo. These cells then were sent to a lab where the single cell from each embryo was tested for the genetic defect in question. We then transferred two embryos pre-determined to be unaffected by the disorder. In October of last year, we welcomed to the world our miraculous bundle of joy, an – unaffected – little boy.

– Name withheld upon request

Preimplantation genetic diagnosis (PGD) is a technique used to identify many inherited diseases. PGD uses DNA amplification to identify embryos with specific mutations of single genes, which may have been acquired from the mother, or father or both.

What PGD can do:
1. PGD can diagnose embryos at risk for some specific genetic diseases if the parent(s) are known to be carriers, and the molecular genetic basis of that disease is known.
2. PGD identification enables elimination of those embryos carrying the genetic mutation that causes the disease in question. It cannot repair those mutations.

What PGD cannot do:
1. PGD cannot guarantee that the baby will be free of all diseases or birth defects because the genetic basis for many defects is unknown. At this time, it is impractical or impossible to screen for most diseases, such as diabetes and cancer, or birth defects such as cleft lip and palate.
2. PGD cannot diagnose all diseases, even if the genetic basis is known, because some of the rarer diseases do not yet have available DNA probes.
3. PGD cannot determine traits, such as eye color, height, intellectual or athletic abilities.
4. PGD is not perfect, despite how sophisticated it is. Errors in diagnosis can occur, albeit at a very low rate. Confirmation of the correct diagnosis should be done by chorionic villus sampling (CVS) or amniocentesis, once the pregnancy is established.

The second type of genetic analysis is what we like to call Preimplantation Genetic Screening (PGS) to look for abnormalities in entire chromosomes missing or extra chromosomes or multiple complex abnormalities in chromosome numbers.

What PGS can do:
1. PGS can screen for abnormalities in 9 out of the 23 chromosome pairs. Currently it is not technically possible to screen for abnormalities in the other 14 chromosome pairs.
2. PGS can help to reduce the risks of miscarriage, commonly due to Monosomy X (one X chromosome) or Trisomy 16 (three of chromosome 16).
3. PGS can help to significantly decrease the risk of Down Syndrome (Trisomy 21) and Trisomy 18, as well as abnormalities in numbers of sex chromosomes (X and Y) (These are among the few abnormalities in fetuses that can survive to the time of amniocentesis and birth).
4. PGS can reduce the number of embryos one must transfer to find the embryos most likely to succeed.
5. PGS may help couples experiencing multiple IVF failures to determine if the failed implantations may be due to aneuploidy (chromosomal abnormalities).
6. PGS can determine the gender of the embryo.

What PGS cannot do:
1. PGS cannot screen for specific genetic diseases couples at risk need PGD.
2. PGS cannot guarantee that the baby will be free of all diseases or birth defects.
3. PGS is not perfect. The detection rate is between 90-93% for the chromosomes analyzed, which is why we still recommend CVS or amnio as a confirmation of PGS findings.

Couples who are at risk of passing on an inherited disease are probably familiar with genetic counselors. However, those who have decided to undergo IVF with Preimplantation Genetic Diagnosis (PGD) will need to see a genetic counselor who is specialized in the procedure of PGD itself. It is important to make sure that the mutation in question can be diagnosed by PGD since not all heritable diseases have DNA probes. And sometimes there are other means of using PGD to determine mutation likelihood.

In cases where there is concern about chromosomal abnormalities rather than single gene defects, Preimplantation Genetic Screening (PGS) is another option that requires a genetic counseling session. The genetic counselor can help patients understand the basics of chromosomes, how they affect the health of embryos and what this testing conveys about the embryos.

Some of the common reasons why patients undergo PGS include:
1. Age (eggs of women >35 years old have a higher risk for chromosome abnormalities),
2. Unexplained recurrent pregnancy loss,
3. Gender selection for genetic disease,
4. History of unexplained unsuccessful IVF cycles.

Who Are Genetic Counselors?
Genetic counselors are health care professionals with graduate degree training in genetics and counseling, and certified by the American Board of Genetic Counseling. Genetic counselors practice in several subspecialty areas of genetics including assisted reproduction technologies, infertility genetics, and prenatal diagnosis. Because your genetic counselor can see you to discuss PGD or PGS, as well as prenatal testing options once you are pregnant, you will receive continuity of care. He/she will help you understand the complex information involved in your PGS cycle, and encourage your own decision making according to your needs. He/she will also serve as a liaison between you, your fertility doctors, and the PGD/PGS laboratory.

What Happens During A Genetic Counseling Visit?
There are two main objectives:
1. Family History Review: Your genetic counselor will take a three generation family tree (pedigree) to identify any additional genetic risks. This process ensures that the type of screening being offered is correct, and to identify any additional testing needed. Medical records may be requested for review.
2. Informed Consent: Informed consent includes an in depth discussion of the PGD/PGS process, from beginning to end, and a review of the information in the consent form, which is designed to inform and protect patients. Important information contained in the consent form includes risks and limitations of PGD/PGS, as well as the purpose of the procedure and the diagnostic technique. Your genetic counselor is available to answer questions regarding its content and to help you thoroughly understand it before signing. For those who have already gone through IVF, the beginning of the IVF with PGD/PGS cycle will be familiar. However, as complex as IVF is, embryo testing adds yet another layer of complexity. Additional steps include biopsy procedures, screening of a single cell for specific chromosome abnormalities or DNA mutations, and reviewing the results prior to embryo transfer. Only a genetic counselor is especially trained to mentally guide you through this process before you are actually in cycle so that, hopefully, there are no unanticipated outcomes.

How Can I Find Out More?
Please contact the Certified Genetic Counselor working with Pacific Fertility Center: Lauri Black at (415) 600-6371.

– Carolyn Givens, MD and Lauri Black, MS, CGC contributed to this article

Several years ago I received a call that would change my life forever. My mother was diagnosed with Huntington’s Disease, an inherited neuropsychiatric disease that affects mind and body – (imagine Parkinson’s and Alzheimer’s combined). The chances of passing on the disease are 50/50, and symptoms usually appear between ages 35 and 50. Because there is no cure, many “at risk” for the disease choose not to learn if they have inherited the HD gene.

Living at risk with HD has altered my life completely. Every choice I make is influenced by the possibility I have inherited the HD gene. And no choice is more affected than that of bringing a child into the world.

As I grappled with my mother’s news, two facts became certain: I wanted children and I did not want to know if I would someday get HD. Given this, my husband and I sought advice on how to have a healthy baby. Our genetics counselor outlined two viable options: Once pregnant I could have a “non-disclosing” CVS that would indicate if the fetus had inherited the chromosome from either my mother or my father, thereby not revealing if I had HD. The second option was a cutting-edge process through IVF called pre-implantation genetic diagnosis (PGD). This involves testing each embryo for the HD gene at the cellular level when the blastocyst is only 5 days old. Only healthy embryos are implanted.

After having experienced a failed pregnancy, CVS had no appeal. That left IVF/PGD. Yet in order to maintain my status as a non-disclosing patient, I arranged NOT to be told any details throughout the IVF cycle. Even knowing how many eggs were harvested or how many embryos were implanted; I could surmise my status. (Imagine: if none of the embryos were healthy, my doctors would stage a fake embryo transfer so I wouldn’t suspect anything.) As such, it was important for all PFC doctors and staff not to reveal any information to me. Doing this meant putting total trust in everyone.

Trying to get pregnant through IVF is a costly endeavor: emotionally, physically and financially. I believed it would be easy because I had gotten pregnant so quickly before. Consequently, I was devastated when our first two attempts failed. In retrospect, I am amazed at how my husband and I endured, despite days when I had almost given up all hope.

After an exhausting six months and three attempts, I was finally pregnant. While overjoyed, I was still hesitant to believe it would go to term. Furthermore, I was required to have an amnio to ensure no errors were made, although given my non-disclosing status, I would not learn the results of that testing. It was only after my fifth month into pregnancy, that I believed I would have a healthy baby.

Our son is a miracle and my husband and I cherish him beyond belief. We are eternally grateful to the people who supported our choice to conceive a healthy baby using PGD.

–Patient’s name withheld upon request

PGD FISH (Fluorescent in situ hybridization) Photo: An embryo with normal chromosomes

We are looking here at the DNA from a cell taken from a human embryo. It has been stained green, and colored fluorescent probes have been applied, which are specific to individual chromosomes. This allows us to count the number of chromosomes and tell if the embryo is normal or not for those chromosomes tested. For the chromosomes that we are interested in counting, we should see 2 brightly colored spots, since we have 2 copies of each chromosome. In this picture, we see 2 red spots (= 2 copies of chromosome 13), 2 yellow (chromosome 22), 2 light blue (chromosome 18), 2 dark blue (chromosome 16) and 2 green (chromosome 21). This embryo has the correct number of these chromosomes. Note that one of the green spots and one of the yellows are very close together in the picture, but they are definitely there.