Since Pacific Fertility Center came into existence in November of 1999, we have been offering genetic pre-screening of IVF embryos for couples with recurrent miscar- riage, repeated IVF implantation failure and sex selection for family balancing. For most of the last decade, a technology known as Fluorescent In-Situ Hybridization, or FISH has been used to screen embryos. FISH is employed to probe a cell removed from a Day 3 embryo to determine the chromosomal makeup for anywhere from three to twelve of the cell’s 23 pairs of chromosomes. With time, we, as well as everyone else in the reproductive genetic world, came to realize the serious limitations of this technology.

In the last 2-3 years, as the Human Genome Project has been completed and as more DNA-related biotechnologies have emerged to evaluate human genes, these methods are being utilized to analyze human embryos. The technology now available—the ability to analyze large numbers of genetic locations on each human chromosome, and quantify that genetic material, with the previously well-established techniques to amplify a single cell’s genetic material up to hundreds of thousands of copies—has allowed PGS to take a quantum leap forward. It is now possible to more accurately analyze all 23 chromosome pairs from a single embryo; not only to determine if the correct number of copies of each chromosome is present, but also to look at single gene mutations.

At the end of 2009, Pacific Fertility Center began working with a new biotech company called Gene Security Network, located in Redwood City (genesecurity.net). This company uses gene microarray technology to analyze amplified DNA from a single cell.

It then uses microchips to analyze 30,000 genetic loci in a quantitative manner. In addition, their unique technology allows us to compare the analysis of the embryos’ cells to the parent’s chromosomes to ensure that all the genes are being properly analyzed. It does appear that the error problems that plagued FISH technology have been overcome with this new, more sophisticated, method.

In October of 2009, Dr. Conaghan and I were invited to tour the GSN laboratory and see the technology in action. We met with David Johnson, the lead scientist at GSN, who explained the cell process; from the amplification of the DNA, to arranging the chromosomes on chips, to DNA analysis, to synthesizing the data generated with the parental genetic data to come up with a full analysis of that cell’s genome. In order to process the cells between the day of embryo biopsy (Day 3) and receive the results on the day of embryo transfer (Day 5), their technicians work around the clock in shifts. GSN has a very cold, clean room to replicate the single cells into multiple copies. They cannot allow any outside contamination, not even from a single cell. They videotape the cell duplicating process so if any errors subsequently arise, they have a video record of what the laboratory technician did. We found this to be very impressive. We also saw how the chips were coated with DNA and analyzed. We were shown the sophisticated software that generates the final report detailing the genetic makeup of each embryo from the cells in which they originated. All in all, the tour gave us great confidence in the quality control and scientific integrity at GSN.

Even with this 21st century technology, we continue to biopsy Day 3 embryos because it provides us with a 48 hours window to send the cells to the lab and complete the analysis in time for transfer. However, we have not yet found a way around the problem of mosa- icism. GSN and microarray technology appears to have largely solved the resolution error problem but it can only tell us what is in the chromosomal make-up of the single cell. It cannot tell us whether or not that cell represents what is truly going on with the rest of the embryo. We are currently looking at the possibility of biopsying Day 5 embryos. The set back would result in having to freeze these embryos due to the time constraint in analyzing the genetic material in time for fresh transfer. With all of the innovation occurring daily in the genetics field, we hope that this puzzle will be resolved.

— Carolyn Givens, M.D.

Previous Fertility Flash articles about PGS:
2 Methods of Gaining Info Prior to Implantation
PGD & PGS: Why Genetic Counseling is a Prerequisite
The Benefits and Pitfalls of PGS

Pacific Fertility Center and The Fertility Flash would like to invite you to a special Valentine’s Day event.
Do You Love Your Genes? Tweetup/Meetup (a Valentine’s Day event)
Thursday February 11, 2010 at 5:30pm
Pacific Fertility Center’s Education Center
55 Francisco St., Suite 550
San Francisco, California 94133 Get Directions

Please join us for genes, love, award-winning wine, chocolate, and tasty, healthy appetizers!

To view the invitation, click here

This is an in-person and virtual event for all who would like to participate and learn about the leading edge of genetics and fertility. We will also be tweeting live during the event to communicate with and connect tweeters.

Genes are an important part of life, especially for those who are struggling to conceive a child.  At this event we will celebrate these building blocks of life in all forms, whether they come from biological parents, birth parents, or donors.

We will also be joined by representatives from Counsyl and the Gene Security Network (GSN) to speak about their cutting edge genetic testing technologies.

For more details on our presenters see:

Pacific Fertility Center: http://pacificfertilitycenter.com
Counsyl: http://counsyl.com
GSN: http://genesecurity.net

**

Please RSVP at rsvp@fertilitywire.com or on Facebook at http://bit.ly/bopZUZ

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—Best regards from all of us at Pacific Fertility Center.

For patient(s) who need to use an egg donor to create or expand their family, medical scrutiny is performed on the chosen egg donor before she can proceed with the IVF cycle.

The medical screening of egg donors is an important process. Here at the PFC Egg Donor Agency, we proceed with an extensive screening process PRIOR to allowing the egg donor to become eligible for choosing by the intended parent(s). This extensive screening is performed to help determine and identify any medical factors which may disqualify the donor, or to identify information which may require additional testing prior to determining donor eligibility. At PFC, our philosophy is that we want to identify any issues prior to intended parents choosing the donor, so that the risk of identifying medical issues with the donor after the start of the IVF cycle is minimized, and the risk of canceling the cycle is much reduced.

Medical screening for the donor includes an extensive review of her personal and family medical history, physical exam and pelvic ultrasound, psychological evaluation (in-person visit with our MFT Peggy Orlin),

standardized personality assessment (PAI), and blood testing including ethnic appropriate genetic testing.

The PFC Egg Donor Agency complies with current recommendations by the American College of Obstetrics and Gynecology (ACOG), and the American College of Medical Genetics (ACMG). The donor identifies her ethnic background, and based on this information, appropriate testing is performed (see article by Lauri Black, Genetic Counselor, outlining current recommendations). This testing is done and results reviewed prior to approving the egg donor as eligible to be in the donor database. If the donor is a carrier for a genetic mutation, this may disqualify her from being an egg donor; some genetic mutations may not be disqualifying, but the sperm source may need to be screened for that mutation, prior to deciding to choose that egg donor. These tested mutations are for recessive disorders, so an embryo would only be at risk of having the disorder if BOTH the egg and sperm source were carriers for the identified mutation (see above noted article).

It is important to understand that new genetic mutations are identified almost every day; so recommendations for ethnic-based testing do potentially change year by year. While many genetic mutations have been identified on the human genome, many of these are very rare, and only mutations that are more frequently seen within one’s ethnic group are those that are recommended to be tested for. It is not appropriate, nor feasible, to check for all known possible mutations. The PFC Egg Donor Agency is kept apprised of current recommendation by our affiliated genetic counselors, so that our list of genetic screening tests may change over time. Rest assured that we keep informed of these changes, and comply with up-to-date recommendations.

While all this testing may seem cumbersome, it is to help assure that once you choose your egg donor, we can proceed with the IVF cycle with minimal risk of a cancellation, and start you on your way to achieve your dream of a healthy family.

Every year, several Pacific Fertility Center professionals participate in ASRM’s national meeting. They evaluate the research and share their findings with PFC and Fertility Flash.

Among those attending the conference from PFC were Dr. Philip Chenette and Dr. Isabelle Ryan and Peggy Orlin, MFT. Their reviews cover the following topics: Update #1: Ovarian Stimulation Techniques, Update #2: PGD and Aneuploidy Screening Techniques, Update #3: Egg Freezing, Update #4: Acupuncture, and Update #5: Men and ART.

Update #2: PGD and Aneuploidy Screening Techniques

Preimplantation genetic diagnosis (PGD) has been one of the hallmark technologies of modern reproductive medicine. The ability to look inside a cell, beyond its visual appearance to the actual genes controlling the cell, has provided insight into the workings of the embryo and a valuable clinical tool to improve fertility care.

The most common use of PGD is to count chromosomes using FISH probes. Using labels that glow under ultraviolet light, a limited number of chromosomes can be identified and counted. Missing or duplicated chromosomes are indicators of abnormalities in the embryo, a condition known as “aneuploidy.” FISH has a significant error rate, and while clinically useful, results must be interpreted with caution.

A new technique discussed at the ASRM meeting is SNP analysis. SNPs are common tags in DNA that can be measured by automated systems. Microarrays of thousands of SNPs have been prepared that provide a clear picture of the chromosome structure of a cell. Microarray-based aneuploidy screening has excellent reliability and accuracy, and holds enormous promise for identifying genetically normal embryos. This study represents the first validated method of analyzing the entire set of chromosomes in a single cell. Stay tuned for more on this exciting technology.

Array CGH uses thousands of very small DNA probes along with computer software to describe the structure of DNA in a single cell. A very sensitive test, it is fast enough to be used during an IVF treatment cycle, and far more accurate than conventional fluorescent probe (FISH) analysis. Array CGH may lead to improved IVF outcomes as embryos containing an error in any chromosome can be detected, which would allow better selection of healthy embryos.

PGD has proven useful for the treatment of recurrent miscarriage. In an analysis of 279 patients with recurrent miscarriage (women who had previously experienced 3-5 miscarriages), researchers in New Jersey found an improved miscarriage rate of 19.5% after PGD versus their 40.9% expected rate.

Philip Chenette, MD

Here are some images from the different techniques that can be used for gender selection

To read more about this process, see our Ask The Experts post

The U.S. marketplace is punctuated with products and services trying to lure desperate parents into believing that somehow, someway, it must be possible to predict and even select the outcome of the baby’s gender through various hocus pocus methods. Perhaps not coincidentally, many products and services, such as www.fortunebaby.com, appear to be subsidiaries of companies based in China and India where male babies are prized over baby girls.

In the line-up of such products, Baby Gender Mentor blood test hit the marketplace with great Public Relations fanfare including a brief interview on the Today Show and a headline in the Boston Globe. Sadly, both of these popular press outlets focused squarely on the debate about gender selection ethics and never seriously questioned the accuracy of such a test. As a result, millions of viewers and readers may have assumed the expensive test results were accurate. Acu-Gen charges $275 to mail order the test.

This was in June. Now, three months later, enough women who were lured into buying the test and assured by the company’s guarantee that it will reimburse misdiagnoses with 200% of their money back, are asserting the test doesn’t work. Many women are trying to get refunds and are being told by Acu-Gen that a “vanishing twin” may have caused the test to fail.

National Public Radio, taking a more critical stand, recently broadcasted a story pointing out that Acu-Gen offers little proof of its claims and admits that it is not required to undergo FDA testing to verify accuracy. On its web site, the company describes how the process purportedly works.

Gender-specific DNA from the fetus floats around in the mother’s blood stream after having crossed over the placental walls. The presence of the Y chromosome in the female blood via a finger-prick blood tests indicates a “male-positive” baby.

A visit to Acu-Gen’s Gender Mentor test web site reveals some other questionable assertions. Men are not allowed to be anywhere near the pregnant woman as she is having her blood drawn for the test. Acu-Gen also lists on its web site the names and publications of noted experts on fetal DNA testing, some whom NPR interviewed and deny any involvement with the company.

The notion that just five weeks into a pregnancy a simple blood test can accomplish what amniocentesis or ultrasound can do much later in a pregnancy is at this point wishful thinking. A dedicated web site: www.in-gender.com takes a more comprehensive and critical look at the claims of many sex-prediction and selection techniques and includes descriptions of the high-tech methods that do work.

– Eldon Schriock, MD

Q.
We recently had PGD performed, and it revealed that two abnormal embryos were developing beautifully and two genetically normal embryos had ceased developing. Why would the genetically normal embryos not develop in comparison to the genetically abnormal embryos? Would this be due to egg quality? Would the same results be expected for a future PGD procedure? Is it unlikely that a six-cell embryo that had not developed in two days would result in a pregnancy?

A.
I don’t have all the information needed to give you a complete answer but I’m going to assume that you are a typical IVF patient (in your late 30′s) and were doing PGD to eliminate embryos with chromosomal abnormalities or aneuploidy. During your IVF cycle the eggs that were harvested from your ovaries were inseminated and those that fertilized and continued to develop were analyzed genetically. Depending on your (maternal) age, somewhere around 50% of your eggs would have been genetically abnormal. The genetically abnormal embryos look and behave in the same way as normal embryos.

Most genetic abnormalities cause an embryo to fail at the time of implantation (5 or 6 days old) or cause a pregnancy to fail early (miscarriage). When we look at embryos under the microscope in the days leading up to transfer, there is no way of knowing which are genetically normal or abnormal. Both types of embryos grow and develop similarly. In fact, some embryos that we know are abnormal (e.g. resulting from an egg that is fertilized by 2 sperm) often develop faster and look more beautiful than normally fertilized embryos.

The egg is a very large cell and when it is released from the ovary it has already been programmed to develop for 3 or more days after fertilization. Mom pre-loads her eggs with the necessary information for this early development. In most cells, including sperm, there are internal checks to make sure that the cell is functioning normally and that it is genetically normal. Cells that are abnormal, commit suicide in a process that we call apoptosis. Eggs however, seem to have a very poor internal surveillance mechanism, and even those that are grossly abnormal (e.g. with a whole extra chromosome) can fertilize and develop even to the point of giving you a live child. Down syndrome is the classic example, although at least 75% of embryos affected with this condition miscarry early in pregnancy.

So, eggs are endowed at ovulation with the necessary information to keep them going and looking normal for days, regardless of their genetic constitution. There is no relationship between their genetic status and how beautiful they look in our petri dish. If there were, we wouldn’t need to do PGD. We can keep embryos alive in the laboratory for 5 or 6 days and some of the abnormal embryos might stop developing by that time. However, our experience with PGD over the years tells us that about 50% of the genetically abnormal embryos will still look beautiful on their 5th day of life.

The pattern of development that we see with human embryos, regardless of their genetic status, is extremely variable. As you have witnessed first hand, normal embryos often arrest for reasons that we don’t always understand. This is true, regardless of whether the embryos are growing inside of you or in our lab, and this leads to a very inefficient process of reproduction in human females because she only ovulates one egg per month. We do know that the younger a woman is, the better the chance that the embryo will continue to grow. Embryos are more likely to fail in older women. In very young women, over 50% of embryos will implant in the uterus, but in women over age 40 less than 10% will implant. Although we can’t fully explain this phenomenon, a major contributing factor is egg age. Since women have all the eggs they will ever have when they are born, a 40-year-old woman is trying to get pregnant with a 40-year-old egg. And 40-year-old eggs just don’t perform as well as younger eggs.

Are PGD results consistent from one cycle to the next? The PGD technicians tell me that they get similar results for a patient 2 out of every 3 times.

Any embryo that has not developed in 2 days will not get you pregnant. If an embryo is to be ready for implantation, it must be alive and increasing its cell number every day. We expect a full round of cell division (e.g. from 4 to 8 cells) every 16 hours. Further, an embryo transferred to your uterus on day 4 or day 5, following your PGD analysis, should have enough cells to begin forming a placenta. It sounds like your embryo had arrested (i.e. it was dead).

Human reproduction is a very complex undertaking, and often patients feel like they’re left with more questions than answers after their fertility treatment. Don’t be afraid to ask your questions, no matter how simple or complicated they might be. Chances are, we’ve encountered your situation before.

– Joe Conaghan, PhD, HCLD

Considering prenatal diagnosis once pregnancy is achieved is an important and complex decision. Although there are a wide variety of screening options available, prenatal diagnosis is the most accurate method for detecting chromosome abnormalities, such as Down syndrome. Diseases like cystic fibrosis, Tay-Sachs, sickle cell anemia, and thalassemias can be tested for if the parents are known to be carriers for these genetic diseases. Because prenatal diagnostic testing allows genetic experts to test placental cells directly, the results are diagnostic and specific for the fetus.

There are two different prenatal diagnostic tests, chorionic villus sampling (CVS) and amniocentesis. CVS is a procedure in which a small amount of tissue (chorionic villi) is obtained from the developing placenta at approximately 10-13 weeks of pregnancy. The tissue is then evaluated for chromosome abnormalities, and if indicated, specific genetic diseases. The primary advantage to CVS is that this test can be performed much earlier in pregnancy than amniocentesis. However, CVS does not detect neural tube defects (spina bifida, meningomyelocele or anencephaly). Therefore, patients who opt to pursue CVS undergo an AFP blood test and a high-resolution ultrasound later in pregnancy to screen for these defects. Also, approximately one percent of all CVS results will show a mixture of normal and abnormal chromosomes, which is called mosaicism. The majority of the fetuses in these pregnancies are normal, however additional testing, including amniocentesis, may be indicated.

CVS can be performed one of two ways depending on the location of the placenta within the uterus. The transcervical method is performed by inserting a thin catheter, guided by ultrasound, through the vagina and cervix to reach the chorionic villi. The transabdominal method is similar to amniocentesis. Using ultrasound, a thin needle is inserted through the mother’s abdominal wall to obtain a small amount of tissue. In either case, this placental tissue is then sent for analysis.

Amniocentesis is typically performed between 16-20 weeks of pregnancy. Under ultrasound guidance, a thin needle is inserted through the mother’s abdominal wall into the amniotic fluid surrounding the fetus. A small amount of fluid is then taken and analyzed for chromosome abnormalities, neural tube defects, and if indicated, specified genetic diseases. The main benefit to amniocentesis is that although it is performed later in pregnancy, it is possible to test for genetic disorders, including chromosome abnormalities and specific genetic diseases, AND neural tube defects, such as spina bifida, all at once.

Whether patients choose CVS or amniocentesis, it is possible to obtain the same information with either procedure. However for patients who choose CVS, it is necessary to do a follow up blood test and detailed ultrasound in the second trimester to rule out neural tube defects. It should be noted that the results from this blood test and ultrasound are not as conclusive on neural tube defects as the results from an amniocentesis. Because both procedures are considered invasive, meaning that it is necessary to enter the womb with either a needle or a catheter in order to obtain cells, there is a small risk of miscarriage due to the procedures. The risk for either CVS or amniocentesis is approximately 1/200. Diagnostic results from either procedure take about ten days to be completed.

Regardless of whether you are considering CVS or amniocentesis, genetic counseling is an important step in your overall decision-making process and in assessing your risk factors for genetic disorders. Genetic counselors are available to discuss in further detail the benefits, limitations, and risks for prenatal diagnostic testing in order for you to make the best decision for you and your family.

– Kendall Glynn, MS, CGC, Certified Genetic Counselor, California Pacific Medical Center

In graph A (pregnant) DNA fragmentation index is nice and low at 7.5%. You can see clearly that there are very few sperm (7.5%) with moderate or high fragmentation and that most of the sperm are bunched tightly together with very little fragmentation. These healthy sperm were able to establish and maintain a pregnancy.

In graph B (not pregnant), the sperm DNA is much more unstable and there is a fairly even spread of low, moderate and high fragmentation. The DNA fragmentation index is 65% and these sperm were unable to establish a viable pregnancy.

Intracytoplasmic sperm injection (ICSI), a procedure where a single sperm is injected into an egg, went into widespread use in the US in the early 1990′s. With it came the view that as long as a man had any sperm, he could father a child. In many ways ICSI was a remarkable procedure, allowing thousands of infertile males to have children. And ICSI worked even when the sperm didn’t swim well, had poor morphology or were surgically recovered from the epididymis or testicle. It appeared as though there was no physical obstacle to fertilization as long as a live sperm was available for injection.

Now, with over 10 years experience with this procedure, and regardless of sperm or egg quality, we understand that on average 70-80% of all eggs will fertilize following ICSI. If we physically place the sperm inside the egg, fertilization happens most of the time. However, fertilization is not a very reliable measure of sperm quality, or even egg quality, and the rate at which your eggs fertilize has little bearing on whether or not your embryos will implant after transfer. Eggs recovered from women aged 40 and older, where we know that egg quality is poor, will fertilize at the same rate as younger eggs. Similarly, sperm with poor morphology will fertilize eggs at the same rate as sperm with normal morphology.

After fertilization, if embryo quality is poor, or if embryos fail to implant after transfer, we tend to implicate the eggs as the likely source of the problem. It is very hard to pin the blame on the sperm and we usually have very little evidence that would implicate the male partner in the failure. After all, much time and effort was needed to get the eggs, the egg is mostly responsible for preimplantation development, and the developing embryo was placed safely in the uterus. The tiny sperm brought only the male’s genetic material or DNA, and we saw that that was safely inside the egg at fertilization.

Even when we start to worry about the DNA, eggs are much better known for genetic problems than sperm. Down syndrome is the classic example, as it is well known that the incidence increases with increasing maternal age. Genetic problems in children due to paternal age are less well known and in fact less than 10% of Down Syndrome cases arise as a result of a genetic error in the sperm.

In trying to visualize what DNA looks like, you have to think of a ladder. DNA is a double strand that is held together by the rungs, and the ladder is twisted and coiled. In sperm or eggs the DNA is organized on 23 distinct structures called chromosomes. Each chromosome is simply a very long twisted and coiled ladder.

When we count chromosomes in sperm and eggs, sperm have the right number about 90% of the time and for eggs this varies according to maternal age. For women over age 40, we would expect at least 50% of their eggs to have an incorrect number of chromosomes. These abnormalities don’t appear to stop eggs from fertilizing, but the majority of the resulting embryos either won’t implant or will miscarry early in pregnancy.

Because we know that sperm don’t carry a lot of chromosomal abnormalities, we have to dig deeper to find problems that may cause infertility. The sperm chromatin structure assay (SCSA) is a test developed to look at the integrity of the DNA. Basically it looks at the structure of the ladder and determines if the strands are coming apart due to broken rungs. The more severe the DNA fragmentation is, the less likely that the sperm can establish a viable pregnancy.

To have the test performed, we ship a frozen semen sample to Donald Evenson, PhD, in Brookings, South Dakota www.scsadiagnostics.com. There the sperm are assessed and any sample with less than 15% DNA fragmentation is considered normal. Levels of fragmentation up to 30% may cause reduced fertility, and men with greater than 30% fragmentation are considered to have significantly reduced potential to father a child.

Environmental stresses such as smoking, exposure to other chemicals or toxins, or any other chemical or physical stresses that the sperm may be subjected to may cause or contribute to high levels of sperm DNA fragmentation. In the testes it takes over 70 days to make each sperm, so the potential for exposure to stress is high. Consequently, it’s important for men to look after their health in the months leading up to their attempts to conceive. As always it’s good to eat well, exercise, avoid illnesses, hot tubs and exposure to toxins and take your vitamins. We particularly recommend vitamins C and E, beta-carotene and anti-oxidants for sperm health. We don’t routinely recommend the SCSA for our male patients since sperm fragmentation is likely to affect a very small number of men. The significance of a high fragmentation index is still under debate as there are reports in scientific literature of pregnancy successes despite a bad test result. Further, it is unclear what the prognosis is for men that succeed in reducing their fragmentation score by taking their vitamins and living healthier lives. An alternative solution for men with high fragmentation is to use donor sperm, however most couples choose to use their own sperm despite high fragmentation.