IN VITRO FERTILIZATION WAKE FOREST UNIVERSITY BAPTIST MEDICAL CENTER WINSTON-SALEM, NORTH CAROLINA Broadcast November 1, :00:13.

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1 IN VITRO FERTILIZATION WAKE FOREST UNIVERSITY BAPTIST MEDICAL CENTER WINSTON-SALEM, NORTH CAROLINA Broadcast November 1, :00: NARRATOR: Denise and Anthony Steele s first child has sickle cell anemia, a chronic illness she inherited from her parents. Having a stroke when she was only 3Ω years old is just one of the medical problems she has faced. 00:00: DENISE STEELE: When you visit the hospital seven times a year with a baby, with an infant, it s no fun, it s no fun. That is really, really difficult, once you ve been through it. 00:00: NARRATOR: Recent innovations in reproductive medicine include a process known as pre-implantation genetic diagnosis or PGD. PGD can identify embryos with genetic defects that cause conditions such as sickle cell anemia, Tay-Sachs disease, and cystic fibrosis. Denise is now pregnant again following in vitro fertilization. With the benefits of PGD, she is looking forward to having a child who does not suffer from sickle cell. Clinics such as the Center for Reproductive Medicine at Wake Forest University Baptist Medical Center are providing patients with the latest techniques of in vitro fertilization, including PGD. During this webcast, the Wake Forest Baptist team will demonstrate these techniques. You may questions to the physicians at any time by clicking the MDirectAccess button on your screen. This program represents the Medical Center s ongoing efforts to bring the latest developments in health care to the community. 00:01: JEFFREY DEATON, MD: I would like to welcome everyone to this hour-long webcast on in vitro fertilization and pre-implantation genetic diagnosis. My name is Dr. Jeff Deaton. I m the Director of the Center for Reproductive Medicine here at Wake Forest Baptist, which is located in Winston-Salem, North Carolina. I would also like to take this opportunity to introduce the other participants in the webcast. On my left is Dr. David Wininger, who is the Lab Director of the IVF lab, and to his left is Dr. Pat Koty, who is the Director of the Genetics Lab at Wake Forest Baptist. 00:02: In vitro fertilization has been a standard technique with infertility treatment for many years, since the late 70s, but the ability to genetically test embryos for specific diseases is a new technique that came into our field about 4-5 years ago. Many of us in the field believe that genetic diagnosis will revolutionize the way we look at in vitro fertilization. Since we have a desire to cover both IVF, or in vitro fertilization, and genetic diagnosis, we have a very diverse audience today. Let me kind of go through what the hour-long webcast will entail. The first minutes will be sort of a general overview of in vitro fertilization. Many of the lab directors and clinicians in the audience will know some of this, but then we will shift for the second half to a discussion about genetic diagnosis. We will bring the Steeles back in at the end of the hour for general questions. We will be fielding questions throughout the entire hour, so if you have a question directed to any of us, please click the button on your screen and we will field those questions during the hour. 00:03: Let s start with just a general overview of in vitro fertilization and you ll see a slide up on the internet. I m going to go through these slides and then we re going to show some videos of all the different procedures we do during this process. Until genetic diagnosis came into the field, in vitro fertilization was done specifically for infertility. As you will see on the first part of the slide, all infertility diagnoses, whether it s male factor, tubal disease, pelvic disease, unexplained infertility, all diagnoses are appropriate for in vitro fertilization. Many couples try simpler treatments first, but all couples eventually become a candidate for in vitro. Now, since we ve been able to do

2 genetic testing, two more types of couples have been coming to see us. The second type are couples where they have had a lot of pregnancy losses, which we call habitual abortions. We have now learned through genetic testing that many of these couples produce embryos with an abnormal number of chromosomes. The third type of couple that come in are couples where they have a specific family history for a genetic disorder, such as sickle cell, which we ll hear more about later, or Tay-Sachs or cystic fibrosis. Now, before we do an in vitro fertilization cycle, we do routine testing on the couple and you see those listed at the bottom of the slide. For example, all women have an evaluation of the uterine cavity, either an ultrasound or an HSG. We do a semen analysis on all the men. There are specific lab tests we do in the females to determine are the eggs good quality? These are done on the third day of her cycle. Then there are other routine tests we do, such as hepatitis, HIV, and rubella testing. It s also a fairly complex process, so our couples spend at least 1-2 hours with us, usually with the nurses, going through the consent process and learning how to give injections. 00:05: Now, before we get into a basic overview of IVF, I do want to cover, in a very simplistic fashion, pre-implantation genetic diagnosis, which we call PGD. There are two basic types of PGD. The first type involves what we call FISH, which is on the top of the slide. It s simply a count of the number of chromosomes. For example, the typical embryo or the normal embryo should have 46 chromosomes, so we just count how many does the embryo have. This is a test for what s called aneuploid, which simply means an abnormal number of chromosomes. We generally test for nine different chromosomes, not all of them, and we ll get into why that is later. The second type of PGD involves single gene defects. This is a process where we look for specific genes in the embryo, such as sickle cell, Tay-Sachs, cystic fibrosis, and many others. We can currently test for over 100 different genes and the number is growing almost daily. 00:06: What are the indications for PGD? For FISH, the main indication currently is advanced maternal age. We have learned that if you have a woman who is over the age of 40, roughly 40-50% of her embryos that look normal are genetically abnormal and we would not know that except for this genetic testing, so one indication is advanced age. A second indication is a history of recurrent pregnancy loss. This is a new indication and we have seen many of these couples now. We ve seen that if we take a couple that has had recurrent pregnancy loss and do genetic testing, often a majority of the embryos are abnormal, regardless of her age. The third indication for FISH technology is a prior failed IVF cycle. In other words, if a couple goes through in vitro, has normal-looking embryos, does not get pregnant, in a repeat cycle we would advocate for genetic testing and we often learn that they have an abnormal number of chromosomes in many of their embryos. The next two indications listed on the slide are sort of newer or I should say in transition. We do believe that there may be a role for FISH technology in cases of severe male factor, and we re also finding that couples are requesting it. We have had several couples who look at me and say, well, Dr. Deaton, I m getting ready to go through in vitro and go through all that effort and spend that money. Can I go ahead and do genetic testing and know that you re putting back normal embryos? That is not an established indication for PGD, but it s one that s being discussed at greater lengths in our field and in our society. The indications for single gene defects are obviously very specific to the genetic disorder in the couple. For example, some couples come in having had a child with a disorder and they would like to have a second child and try to avoid that disorder. Other couples come in with simply a family history. They don t have any children, but they know their family has a genetic disorder that they would like to screen for. 00:08: Now, when we get ready for PGD, we have a setup at Wake Forest Baptist where we run a combined Tuesday afternoon clinic where couples can come in, speak with one of the physicians, and also speak with our genetic counselors. We feel that all of these couples need genetic counseling. We also have a separate consent process for the in vitro and for the PGD. We don t simply put it in one process. We feel that it s complicated and we want couples to understand what they re doing regarding PGD. Dr. Koty, which you will meet later or get to hear from later, often needs blood from the couple, from both the husband and the wife, to run testing prior to the cycle and we can obtain that blood at the same time we do the genetic counseling. Finally, we have an ethics committee at Wake Forest Baptist that has been very helpful in deciding which disorders we should be testing for and actually which disorders we should not be testing for. They have put in guidelines for us and if we feel that there is a couple that is requesting testing that is sort of in a gray zone, so to speak, we will actually consult with the ethics committee. 00:09:59.000

3 Now let me run through the timeline of IVF and then we re going to show you a video of an actual egg retrieval. We use what s called a long protocol in most of our couples, which means we give them either Lupron or Synarel, to put them into a brief medical menopause. That allows us to control the cycle. Following that medication, they begin a twice a day injection of gonadotropins, which is the fertility shot. Gonadotropins are what go to the ovaries and develop eggs. During the time they re on gonadotropins, and it s about a 10-day course, we do several ultrasounds where we monitor their egg response in their ovaries. Once the eggs are ready, and we have certain criteria to determine maturity in eggs, we do the egg retrieval, which you ll see in a minute. Once the eggs are removed, at the time of the egg retrieval, Dr. Wininger makes a decision about whether they will be fertilized in a conventional way or injected with sperm. Then, if we re doing PGD, we develop the embryos to the day 3 stage and to the biopsy. Then, once we get the results back from Dr. Koty s lab, we decide which embryos to transfer and do the final embryo transfer. 00:11: What we re going to do right now is take a few minutes to show you an actual egg retrieval. You ll see that the egg retrieval room is adjacent to our lab. That is the pass-through window where the nurse will transfer the samples into the lab. That is obviously me getting ready to do a patient. These are done under IV sedation. Patients are very comfortable, but they re not asleep. Some of them talk during the procedure, but after it s over, they all say they were very comfortable. This is the needle, on ultrasound, going into the follicle. This is the follicle which has the egg attached to the wall. The needle takes the fluid out and then once I finish with one follicle, I sort of back up, redirect the ultrasound probe, and go into the next follicle. Notice that the ovary, which is right here, sits adjacent to the vagina, so it s very easy to get into the ovary with the needle. Again, we go into a second follicle. We remove that fluid and then usually after 2-3 follicles, the tube is full of fluid, so the nurse changes the tube out, caps the tube, and transfers it to the lab. 00:12: DAVID WININGER, MD: At this point, I pick up the tube and pour it out in a Petri dish under the microscope and locate the oocytes and we continue doing that. This is a warm surface here under the hood, under the microscope. We pour the fluid out in the Petri dishes, locate the eggs until all the eggs are found. 00:13: JEFFREY DEATON, MD: And I keep going. We don t wait for the lab. They re pretty fast, but we keep going, so as Dr. Wininger s looking for the eggs, I m into some more follicles. So we repeat this process, obviously, over and over. If there are only a couple of legs, it takes literally five minutes. If there are a lot of eggs, and I think the most we retrieved has been about 55, then it takes a little bit longer, but the longest retrieval tends to be never more than about 25 or 30 minutes. Again, the nurse simply keeps changing the tube as it fills up with fluid and transfers it to the lab. 00:13: DAVID WININGER, PhD: And I continue to get the tubes, pour them out into Petri dishes under the dissecting microscope, and locate the oocytes. There are a lot of different cell types in the fluid from the ovaries, so sometimes there are cells that don t contain the eggs, so we continue doing this process until all of the follicles are gone. 00:14: JEFFREY DEATON, MD: Now I ve gone to the other ovary, which you can see a lot of follicles in this ovary. We use a single lumen needle, so we do not flush fluid back into the ovary. We simply go from follicle to follicle, which is why it can be fairly quick. Then Cathy or the nurse keeps transferring the fluid into the lab. 00:14: DAVID WININGER, PhD: And I just continue until all the tubes are gone. Most of the time we stay caught up, pouring the tubes out, and we locate the oocytes in the fluid, move them from one solution to another solution, rinse the cells, rinse the egg to remove blood and other cell types until all the follicles have been emptied. Again, all the surfaces we work on when the eggs are out of the body and out of the incubator, all of them are 37oC, which is body temperature. Here, this is what I see under the dissecting microscope. The egg retrieval is over at this point. We have all of these egg cumulus cell complexes and I ll point out to you what I m talking about here

4 in a minute. At this point I m trimming all of the excess cells off of the oocytes. The oocyte is inside this group of cells. A lot of cells are granulosus cells, cumulus cells. These are the cells that produce the estradiol and progesterone that we monitor during the stimulation process, so we cut most of those off. The egg itself is just a little dark spot inside the group of cells. I take all of these cells or most of these cells off prior to putting the cells and the eggs into the incubator in individual drops. So you can see an egg here that just has a few cells around it. You can see a dark spot in the middle. That is the oocyte. Then there s another complex here that has a large group of cells around it. So I just try to trim most of these cells off in preparation for either doing a conventional insemination or the ICSI insemination. So sometimes this may take a while, depending, like Dr. Deaton said, sometimes we may get two eggs, sometimes we may get 50 eggs. So this can take a while to go through this process, removing all the cells. This is not a very high powered microscope. We ll be looking at the oocytes better when we get ready to do the ICSI process, when we can tell actually if the oocytes are mature or not. These are what the eggs look like after we trim the cells off of them. Just a few cells left around the eggs. While this is going on, another technician is preparing the semen sample for the insemination. This will include a sperm count, motility, sort of doing a basic semen analysis. Generally we know ahead of time whether a patient s going to have conventional in vitro fertilization or whether intercytoplasmic sperm injection, or ICSI, is required, but occasionally the sperm count will be different than we would expect, so we have the ability to go ahead and do the ICSI process if we need to. This is just a tube of the separation medium we use for the sperm. We use a density gradient solution to separate out the nonmotile sperm from the motile sperm and put it in the centrifuge and spin out all of the cells that we don t want. 00:18: JEFFREY DEATON, MD: Once we have obtained all the eggs, it takes, like I said, roughly 20 minutes, at most, to get the eggs out. The medicines used for egg retrieval are very short-acting, so most patients go home within the hour and are back to work or full activity the next day. They do take some Tylenol, but they don t usually need anything stronger than that. 00:18: At this point I d like Dr. Wininger to kind of go through what happens with the egg and the embryos for the next several days. 00:18: DAVID WININGER, PhD: Following the eggs retrieval, again, we decide whether we need to do ICSI or conventional insemination. We generally know this ahead of time. So just after the egg retrieval, the eggs go into the incubator. This is a picture of a mature egg at retrieval. Again, this is under a different microscope, where we can really look at the egg. The egg is just the round structure in the middle. There are a lot of cells that surround the egg and these are the cells that we would remove prior to the ICSI procedure. Again, we do about 70% ICSI, based on male factor cases and also if we re doing PGD for single gene defects, we have to do ICSI for those cases also because we re evaluating the DNA and the sperm has DNA and we don t want to have any contamination during the PCR process, so if we re doing ICSI, all those cells that surround the egg, we have to remove those cells with a chemical called hyaluronidase, so we mix the eggs with this chemical, remove all the cells to determine which eggs are mature, and by mature, they have to be at a certain stage of myosis, meta phase 2, and I ll show you a picture of that in a minute, but we can only inject the eggs that are mature. I will show you a video of ICSI next. I mention here that we can use epididymal or testicular sperm. Sometimes men that have had vasectomies or have a problem called congenital absence of the vas deferens, were sperm can t be released, well, we have a reproductive urologist on our staff that can do a surgical sperm retrieval and we can do this ahead of time in most cases and freeze the testicular tissue for use when the female comes through her IVF cycle. 00:21: This is a mature meta phase 2 oocyte. By mature, it means it has this structure we look for. That s called the first polar body. That tells us that the oocyte is at meta phase 2. It can be fertilized at this point. You can see this egg, all the cells are gone now, so this looks totally different than the egg after retrieval. 00:21: This is just a still picture of ICSI, where we have the polar body up out of the way. The chromosomes are situated near the polar body, so we generally try to get the polar body at 12:00 or 6:00. This is all done under a

5 more high powered inverted microscope with joysticks that hold these tools. This is a glass pipette, a holding pipette that holds the egg. This is the injection pipette where I would pick up the sperm. I ll show you now the video of the ICSI process. 00:22: This is the inverted microscope I was talking about and this is a very dilute sample of sperm that s in a viscous solution called PVP, so we get just a few sperm in this solution so slow it down. Then you actually have to nick the tail of the sperm before you inject it into the egg and this activates the sperm prior to injecting it. You don t cut the tail off. Sometimes people see this and say why are you cutting the tail off? Really, you re just nicking it and getting it ready for injection. Again, the eggs are in a little Petri dish up here on the microscope stage, again which is warm. Here are the sperm being immobilized and pulled up into the ICSI pipette. I generally pull 3 or 4 sperm into the pipette and then move into a separate drop. There will be other drops that will have the eggs in them and we ll go to the egg drop and inject sperm. So here you see the joystick apparatus here. This is the oocyte. We penetrate the shell or the zona pellucida, go into the cytoplasm of the egg, mix a little bit of the cytoplasm with the sperm, and inject it back into the center of the egg. Then we ll go to the next egg. Again, here s the polar body here, the shell, the zona pellucida around the oocyte. Here comes the ICSI pipette. The sperm is in the tip. It s mixed with some cytoplasm and injected back into the center of the egg, so the sperm is in the center there. This just continues until I ve picked up enough sperm for all of the eggs. This is just like we re trimming the eggs off at the end of the retrieval; the more eggs you have, the longer this process takes. It can be from 1 egg to mature eggs. Also, it can take varying amounts of time to find the sperm as well, depending on whether it s a testicular sample of a regular ejaculated sample. Generally it takes seconds per egg to do ICSI. After we re finished, the injected eggs are put back into individual drops under oil. This is an insemination media we use and the eggs and Petri dish are put back in the incubator at body temperature and 5-6% carbon dioxide. 00:25: JEFFREY DEATON, MD: Thanks, Dr. Wininger. Of course, this has been a big day for the couple. They have gone through all of the medicines, egg retrieval, and Dr. Wininger has fertilized the eggs, so it s kind of a natural break point, so why don t we take a couple of questions now. The first question must be from a couple. It says I m 32 years old and have been trying to get pregnant for one year. Should I go see a reproductive specialist or my gynecologist at this point? That s a very good question. It s a common question I get from a lot of couples. Many infertility diagnoses are fairly simple to treat. Some of the women that have irregular cycles, it doesn t take much in terms of fertility drugs to get them pregnant, so we feel it s always good to start with your gynecologist and see if they can come up with maybe what s the problem and then refer you. Most gynecologists are pretty quick to refer to a reproductive specialist, like at Wake Forest Baptist if they encounter something complicated or difficult. Obviously any couple going through in vitro or the high tech infertility treatments should be in the case of a reproductive specialist. 00:26: The other question, which is sort of tied into it, isöand this we can both addressöwhat happens during IVF if the female eggs do not mature as expected? I m sure Dr. Wininger would agree that that s probably mostly my job. One of the reasons that we go through extra training in reproductive medicine is to learn these fairly complicated ovulation induction protocols, so if we do our job correctly, then I will give him a mature number of eggs. We expect 80-90% of our eggs to be mature and if they re not, we look at our medication protocols, primarily. Dr. Wininger, is there anything you can do if you get mostly immature eggs? 00:27: DAVID WININGER, PhD: There are techniques we can do. In vitro maturation is something that a lot of talk is going on about. Sometimes we can put the immature eggs in culture and they may mature in two hours or sometimes overnight and we can inject them. Generally fertilization is not as good with those eggs at this point, but it is something that a lot of active research is occurring on. 00:27: JEFFREY DEATON, MD: At this point, Dr. Wininger, why don t you take us through the new few days and lead us up to the embryo biopsy? 00:27:45.000

6 DAVID WININGER, PhD: After we do the fertilization process, either through ICSI or conventional fertilization, where we just mix the sperm with the eggs, about 16 hours after that, we would look at the embryos under the inverted microscope again to look for fertilization. We look for these structuresöthere s two structures inside the egg called pronuclei. There s two structures here. You see two round structures. One is the DNA or the nucleus of the sperm and one is the DNA or the nucleus of the egg. This is what we like to see. We call these pronuclear embryos. Occasionally we ll see two, three, four, sometimes one, and those are abnormal fertilized embryos and we generally don t use those embryos. This is, again, a pronuclear embryo and we would transfer these embryos to a different type of culture solution at this point. The next day, the embryo should be at the 2-4 cell stage. This is a 4-cell embryo and this is a nice, even 4-cell embryo. We would just look at this briefly, just to see that embryos are making progress. The next day, the embryos should be between 6 and 8 cells. About half of our patients have transfers at this point, at the day 3 stage, embryos about 72 hours old. If they have PGD or if we can go to blastocyst, which the other half of our patients go to blastocyst, so we can reduce the number of embryos we transfer, have a better pregnancy rate, we feel, at the blastocyst stage, but again if we re going to do pre-implantation genetic diagnosis, which would be the stage where we would do that. We would go in and take one cell out of the 8-cell embryo for genetic testing and then we would send it to the genetics lab. 00:29: Now, the biopsy procedure itself, we like the embryos to be between six cells and eight cells or better to do the embryo biopsy because, again, these embryos have to make it up to the blastocyst stage, so you could biopsy a 4-cell embryo if you wanted to, but the chances that it would be a normal embryo and also make it to the blastocyst stage aren t very good, so we try to take all the 6-cell or better embryos, put them in a special media that does not have calcium or magnesium, and that loosens up the junctions between the cells, so we don t damage the other cells when we remove one. A hole is made in the opening or the shell of the embryo with a laser because we have to access the cell or the blastomere, so we use a laser to make a hole in the zona pellucida to get to the cell. We try to look for cells that have a visible nucleus because if they don t have a nucleus, they don t have the DNA and it won t be any use to the genetics lab if we give them a cell that doesn t have a nucleus. For aneuploidy testing, the cells that are going to have FISH, we take those cells and fix them on microscope slides with a fixative and we would do, if I biopsied 10 embryos, I would fix 10 cells on these microscope slides. If it s going to be single gene testing, I would put them in a special lysis butter the genetics lab gives me, that lyses the actual cell and just leaves the nucleus intact. 00:31: So now we re going to show the video of the biopsy procedure itself. This is the lacer procedure at the little red arrow. This is where the laser is coming out of the lens out of the microscope, so I d look at it, line up the target with this pointer, and push a button. You can see in this embryo there s a large gap in the shell or the zona pellucida. This allows me to move this embryo biopsy pipette into the interior of the embryo and get a cell. You can see the nucleus, a visible nucleus in this cell, so I try to do the laser hatching at a point where there s a cell that looks good, that has a visible nucleus. Suction is put on this embryo biopsy pipette very slowly, so we don t lyse this cell, and we pull it up in the biopsy pipette and then I will put it back into the culture dish here. I will continue doing this on all of the embryos that I need to biopsy. This is what the embryo looks like after the procedure. You can just kind of see a little gap here where the cell was taken out. Here s the cell with the nucleus in the middle of the cell. That is what we re going for. This process is not detrimental to the embryo at all. Again, that s why we like to do them on embryos that are at least the 6-cell stage. 00:33: JEFFREY DEATON, MD: Thanks, Dr. Wininger. Like I said at the start, until 4-5 years ago, this is where in vitro sort of stopped. We put them back and we did a pregnancy test. We are now able to do the genetic testing, which again is revolutionizing our field. We do have one more question. We do appreciate your questions, by the way. Again, you can them any time during the broadcast. This question is what is the general cost range for in vitro fertilization and does insurance ever cover it? We could talk a lot about that question, but let me try and give you a quick answer. The general cost range for the non-medical, non-medicine portion, so the procedures, is usually around $7,500-8,000 for the basic IVF cycle. The medicines are about $3,000, so the total medicine plus procedures is in the $10,000-11,000 range. Insurance is variable, based on who you work for. Many companies

7 do cover it in our area and we re trying to encourage other companies in our area to cover it, but it s basically dependent on where you live and your employer. 00:34: At this time, though, we need to shift gears and talk about the more complex portion of the webcast. That s the actual genetic diagnosis. I d like to turn that part over to Dr. Pat Koty to kind of go through what he does now that he has the cells from the embryo. 00:34: PATRICK KOTY, PhD: Thank you, Dr. Deaton. I d first like to go over a video, very quickly, that gives you an overview of how we process, first, a FISH PGD slice, so if we can go to that. Here we re getting the slides in. They re already fixed. They ve been biopsied from the embryos. We need to add the probes in order to detect the chromosomes that we want to look at, the nine chromosomes, so here we see it being physically added to the slide that has the cells on it. We add a cover slip so this mixture doesn t dry out because we need to incubate this at a 37o temperature usually for 2-3 hours, sometimes overnight. Again, we want these probes to physically attach to the DNA within the cells so we ll be able to basically do a count of the chromosomes. So here you see that after they ve incubated or hybridized for 2-3 hours. We then wash any excess probe. Then we look under a fluorescent microscope in order to visualize these probes, again labeled with different colors, so we use different filters, as you can see, to fluoresce those probes. Once we get those under the microscope, we would then take an image of these cells with the probes lighting up. You see that here on the computer screen. We basically count the number of red spots or green spots or blue spots, depending on which chromosomes we re looking at at that point in time. 00:36: Next I want to go over, basically, again, the requirements that Dr. Deaton indicated for aneuploid FISH. We need to have informed consent. We also recommend a chromosome analysis be done on the parents, if they haven t been done before, because again, they may be an unknown carrier for a balance translocation. We also would like the IVF center to notify the lab of an anticipated start of the cycle so we can be prepared for it and have the proper staffing in place. For inherited balance translocations, what we need is informed consent again, but in this particular example we would need peripheral blood from the carrier of the translocation at least 6-8 weeks prior to starting the IVF cycle. We would need to confirm the arrangement in the parent who carries it. We would then develop and validate a family-specific set of probes that can identify this particular rearrangement. We would then notify the IVF center that pre-testing has been completed and they can start the cycle. 00:37: Next I d like to go and take a better look at the actual techniques of fluorescent in situ hybridization. So if we can look at the slide a little bit closer, what we see here is these are the probes that we would use. Again, they are specific for a chromosome that we re interested in identifying. These probes are labeled with a fluorescent dye, which again we ll visualize under the microscope, but what we need to first do is denature both the cellular DNA that are on the slide to make it single stranded, instead of double stranded, and denature the probes, allow them to again anneal or hybridize for a 2-3 hour period, and then at that point we look under the microscope and we should be able to have a clear signal on a particular chromosome and interface, and we would just count the chromosomes. There are some labs that only offer 5-probe sets, which are able to detect approximately 43% of the aneuploids in an embryo. We and other labs offer also 9-probe sets, which is able to detect about 79% of the aneuploids you would see in an embryo. Again, if we can take a look at the actual hybridization a little bit closer, this is an example of the first hybridization we would do. This include five probes. We cannot do all nine probes at one time, so we do five and then four. Here we have chromosomes 13, 21, 16, 18, and 22. What we would ideally like is just basically to see nice clean signals, either 1, 2, 3, so on and so forth. Oftentimes we get these additional signals that you need to be able to identify and translate into whether it s a true signal or just background, so that s what we do, again, using the microscope and the computer. What we see here is two 13s, two 21s, two 16s, two 18s, two 22s, so at this point in time, with the first hybridization, we would consider this a normal embryo. Again, what we would need to do, then, is to strip these probes off and then rehybridize with the second set of four probes. Again, what we see here is we re looking at chromosomes 15X, 17, and Y. We do the same thing. We identify which is a true signal, which isn t true signal, and at this point in time, this embryo would be considered a normal female embryo and we would suggest it be transferred, if at all possible. 00:40:05.000

8 Now, what we also do is then report to Dr. Deaton on all the embryos, so we have embryos listed here and what we would ideally want to see is for chromosomes to have just two signals. We would consider that normal. If we re seeing missing chromosomes, such as 1, or additional chromosomes, such as 3, we would consider that an abnormal embryo and we would require that it wouldn t be transferred. Now, you can also see at the top of this slide an area where we indicate no cells present. That could happen if the embryo, the biopsied cell, didn t have a nucleus in it, where we wouldn t detect any signal, but also we can have either a partial or an incomplete hybridization, where we wouldn t get signals for every chromosome we re looking at. 00:41: Now, what s important, if you noticed from that report, there were a lot of abnormal embryos. This is because there s a much higher frequency of aneuploid embryos as compared to live-born babies, so again we ll be reporting out a lot of abnormals. Now, there also is a risk of misdiagnosis associated with PGD testing. For FISH, we quote a 10-15% misdiagnosis risk, based on what s out in the literature. This is due to poor hybridization. You can get false positive or negative. Also a mosaic embryo, where you not only have normal cells within that developing embryo, but you have abnormal cells within that same developing embryo, so again, we re biopsying only a single cell. So if we pull out a normal one and analyze it, we re going to interpret it as being normal, when in actuality the embryo may have a large majority of abnormal cells. So this is why we would advise also to confirm this PGD testing with prenatal testing. Now, I d next like to go, again, to a video to give you an overview of single gene testing done in the lab, how it s processed. Again, we get these biopsy cells in a tube for single gene testing. Here we add all the reagents. The lysis buffer basically bursts the cell, allows us access to the DNA inside those cells, so these are reagents that are necessary for the PCR amplification. I ll go into that in a little bit more detail. We do it under a hood to minimize external combination of the cells, DNA, previous amplification products. We then take it to another lab, where we actually do the physical amplification in a PCR machine. You see that here. After that s been done, we then take it to a genetic analyzer, where we actually visualize the PCR products to determine whether we have normal or mutants that we re screening for. Here you see it s done through a computer-generated readout that we look at and see again whether the embryos themselves are normal or abnormal. 00:43: So if we can look at a slide of the PCR chain reaction, again, every single gene testing requires PCR to be performed. It s a multi-step process. We have steps which include denaturization of the cellular DNA. We have to make it, again, single stranded. We have another step, annealing process, in which we lower the temperature to allow the primers to hybridize to the cellular DNA. Again, we re only interested in amplifying the particular product that we re interested in, the specific gene that we re looking at. Then we also increase the temperature to 72oC, in which we allow the enzymes, the DNA polymerase, to amplify the region within these primers to get additional product. You see that in this slide, where we re able to go from a single cell, just two copies of a particular gene we re interested in testing, to after 36 cycles, which is typical, to 68 billion copies of that particular region we want to test. That s a very powerful tool that we have now. 00:44: If we can again look at the printout of this particular type of testing, we test for single disease status. We also test for markers and I ll go into what markers we use and why we use them, but we see here, with disease status, we see dad has one peak. This is signal intensity. This is separating based on size. So dad, here, has one peak for the disease we re testing for. He s normal. On the other hand, mom, here, we see has 2 peaks, one normal one, one mutant peak, so she s a carrier of the particular mutation that we want to identify. Now, if we go to embryo 1, we see, again, one normal peak, one abnormal peak, so this we would consider an abnormal embryo. On the other hand, with embryo 5 here, we see one normal peak, no abnormal peaks, so at this point in time we would still consider it normal. 00:45: Now, if we go to the next slide, where we re looking at the marker, we use a high polymorphic marker to be able to tell whether we re amplifying the embryo or potentially any contaminant that is in the tube that you did not want in the tube. So with dad we see he has a particular genotype of A and D. Mom has a different genotype of B and C. Now, what we need to be able to see in the embryos is one genotype, either A or D, coming from dad, and the other genotype, either B or C, coming from mom. So in embryo 1, we see that embryo has C and D, so again, one from each, so we know we re amplifying the embryo s DNA. In embryo 5, again, this is the normal one by disease status. We see one a little A and one a little C, so again we re identifying one from dad and one from

9 mom. So in that case, we again know we re amplifying the DNA from the embryo and we would suggest to implant that particular embryo. Now, we would then send a report listing all the embryos, the status of the disease of the embryo, and the marker status, so we have a scenario where you could have normal formation and again we could identify both markers in the embryo, one from mom, one from dad. We could have a situation where we have a mutant disease status, again informative, or we could have no amplification in either disease or in the marker. That would indicate either the cell didn t get properly put into the tube or wasn t properly lysed, so we don t get any amplification whatsoever. Also, we can have partial amplification if we have disease status being identified but no amplification of marker, so that is limited information. We still aren t sure whether we re amplifying the embryo or not. Again, we can have a last scenario where it s amplifying disease status, you can have normal but a contaminant present when we look at the markers. Now, this single gene is also associated with a misdiagnosis risk of 5-10%, again based on what s published in the literature. This is due to a little dropout. Again, no amplification of what you re looking at gives you a false positive or negative and we again advise to confirm with prenatal testing. 00:48: Now, finally we go to the Steele family. They came to us requesting sickle cell testing, PGD testing, because they had an affected child, Niya, who has sickle cell disease. In addition to that, they would have liked us to test for HLA genotyping to potentially match a future child with their current affected child for potential future core blood stem cell transplant or a bone marrow transplant, so we not only had to identify the mutations in the parents and in Niya. We then looked at four polymorphic markers, informative markers, not necessarily for contamination reasons at this point in time, but to genotype for a potential HLA match, so we used markers to expand the HLA regions AB and DR, so hopefully we can have a genotype match. We developed and validated the assay. One thing of note that s important: if we were just looking at disease status of the embryos, a disease-free embryo, out of every 4 embryos, 3 of them should be statistically disease-free, but when we add disease status or disease-free and an HLA match to Niya, if we had 16 embryos, statistically only 3 of those would be disease-free and an HLA match. What we received in the laboratory was 4 embryos to be tested. We identified 3 of those to be disease-free, so we hit the statistics right on the money, but unfortunately, we were unable to find any of them that matched Niya for future potential HLA genotyping. So at this point in time, again, we would give this information to Dr. Deaton and Dr. Wininger for transfer of the embryos. 00:50: JEFFREY DEATON, MD: Thanks, Dr. Koty. We continue to get some great questions. I ve got one for you, Dr. Koty. This one is from an IVF lab director. If we want to request both aneuploidy and single gene testing on the same embryosöi guess if you had a 39-year-old woman who also has sickle cell, for example, or Tay-Sachs or something, would you need one cell or two cells from the embryo? 00:50: PATRICK KOTY, PhD: The way technology is today, we would need two cells from the embryo because technologically we can t do FISH aneuploidy and single gene on the same cell. In the future, hopefully we will be able to offer that. It s in development right now, but currently we would need two cells. 00:50: JEFFREY DEATON, MD: As you might imagine, this is sort of the culmination of the whole process. The couple has gone through multiple injections, an egg retrieval, Dr. Wininger has done the fertilization, we ve done the embryo biopsy, gotten the cells to Dr. Koty, he s gotten the report back to us regarding which embryos are normal and which one are not normal. Now we come to the actual transfer. For the couple, this really is the culmination of the whole process and it can be a very emotional day, hopefully emotional in a good way, but it s a big day for the couple. Dr. Wininger, why don t you address the question we just got from another viewer. This one asks what are the risks of multiple births and how can you reduce that risk? I think it s pertinent to the embryo transfer day. 00:51: DAVID WININGER, PhD: We know the more embryos we transfer, the higher the probability of multiple births. High order multiple births, three or more, we know can be dangerous to both the babies and the mother, so we try to reduce the number of embryos we transfer. What we do now, when we can, is go to day 5, the blastocyst day, where we can reduce the number of embryos we transfer to reduce multiple pregnancies while giving a very

10 good chance of pregnancy. Our pregnancy rate with blastocysts is better than our day 3 transfer while transferring fewer embryos. We still have a high incidence of twins when we do blastocyst transfer, but so far we haven t had triplets when transferring two blastocysts. 00:52: JEFFREY DEATON, MD: Great. Why don t you walk us through the embryo development for the next several days and how we get to the actual transfer day. 00:52: DAVID WININGER, PhD: The next day after the embryo biopsy would be day 4 of embryo development. It would be the morula stage, so we don t do anything with the embryo at this point. The embryo at the morula stage, all the cells start to merge together. It looks a lot different than the embryos prior to this point. You really can t differentiate one cell from another, but this is a nice-looking embryo. It all just kind of molds together into one group of cells. Day 5 is the blastocyst stage. This is where we would do the blastocyst transfer on patients that we re just taking to blastocyst to reduce the number of embryos to transfer or for patients that have had PGD. So at this point we would get the results back from Dr. Koty, telling us which embryos to transfer. Up to this point, all embryos are cultured in individual drops, so we can go through Dr. Koty s report, see which number of embryo is normal, which is abnormal, and then we would look at the normal ones and hopefully the normal embryos would be these nice-looking blastocyst embryos. 00:53: This is another picture of a hatched blastocyst. You can see the zona or the shell of the embryo up here on the right and the intercell mass of the blastocyst is here. This will become the fetus or the baby. Differentiation has occurred at this point, so you do have a part of the embryo that will become the baby. The rest of these cells, the trophectoderm cells will be the placenta, the amnion, the chorion, the extra-embryonic membranes that will actually implant in the uterus. As I said, hopefully at this point we will have nice embryos that are also normal. Just because embryos make it to blastocysts, they re not always normal, so our patients that go to blastocysts that don t have PGD, we can t guarantee them that the embryos we re transferring are no aneuploid because we do know that a lot of aneuploid embryos can make it to this stage. So this would be the point that we would get to the embryo transfer. 00:54: JEFFREY DEATON, MD: At the time of the embryo transfer, we bring the couples back into our conference room. We actually show them their embryos, look at the report, and make a decision how many to transfer. Like Dr. Wininger said a while ago, we primarily transfer two blastocysts, especially in a woman under the age of 37. In older women, 38 and older, we often will do an extra embryo put back. In terms of embryo transfer, the slide here goes through the basic process. We select the normal embryos from Dr. Koty s report. We try to put back two in a younger person, but we have rare, rare triplets over the age of 36. So in a woman who s over 36-37, we don t worry as much about triplets or anything higher. We still get some occasional twins. Embryos are transferred to the uterus under ultrasound guidance and the embryos are placed roughly 2 cm from the top of the uterus. I ll show you one of these in a minute. We use a standard Wallace catheter, which is very atraumatic, very soft, in a majority of our patients. If we have more than 2-3 embryos that are going to be transferred, we can freeze the extra embryos for later use. This is nice for those couples who want to come back, you know, years later, for a second child, for example. Then, once we ve done the transfer, the woman lies there for roughly 30 minutes. Then, after that, she goes back to normal activity. We don t believe activity plays a role in who gets pregnant. We do a pregnancy test and a progesterone level because she s on extra progesterone support during this time. We do those tests roughly 13 days from the egg retrieval or 8-9 days from the actual embryo transfer. At this point we want to show you actually the process that s involved in an embryo transfer. 00:56: DAVID WININGER, PhD: Here I m preparing the embryos and the catheters for the transfer. We re loading, rinsing out the catheter, the syringes. The catheter is basically a long plastic tube. We put a 1 cc syringe on the end of the catheter, rinse it with culture solution, and the embryos are again in a single drop, under oil, on this warm microscope stage, so I m getting ready here. I m still rinsing the catheter. Dr. Deaton during all this is preparing the patient for transfer, washing her cervix, doing an ultrasound, checking out the uterine depth once again. Here I m putting the catheter under the oil, into the microdrop that has the embryos in them. I ll load the embryos in about 20 microliters of the culture media, which is a very small amount, then I ll take the catheter in to Dr. Deaton, who will do the transfer under ultrasound guidance.

11 00:58: JEFFREY DEATON, MD: Notice our in vitro fertilization lab is adjacent to our embryo transfer room. We do this under ultrasound guidance. Robin, one of our lab techs, also is superb at running the ultrasound machine, so as I place the catheter through her cervix, we re actually able to watch it on the ultrasound machine. The catheter has an echo tip, so you can actually see the tip of the catheter right there, starting to track into her uterine cavity. Notice it s coming into the body of the uterus. It stops about 2 cm short of the top. There s the top right there. There s the tip of the catheter. There s the uterus. There s a full bladder so we can see the ultrasound image. Once we get the catheter to the right place, Dr. Wininger hits the plunger, which injects the embryos into the uterus right about there. Then I slowly take out the catheter. I don t like to take it out quickly. I remove it fairly slowly. Most women have a slight sensation when the catheter goes in, but there s no pain involved with this procedure. This is pretty much of a painless procedure for the vast majority of the women. It s obviously emotionally climactic. It s the day they ve been waiting for, but it s a fairly simple procedure in terms of what we have to go through. Once we re done with the transfer, she lies there for 30 minutes and then we let her empty her bladder and go about normal activity. We have not seen any change in pregnancy rates based on whether she lies there for 30 minutes or four hours. Many of the practitioners listening will remember that in the older days of IVF, they would often lie there for 4-6 hours. Now we ve learned that does not matter and they go about normal activity pretty quickly. Dr. Wininger is here, checking the catheter to make sure it s empty because every now and then a catheter will retain one of the embryos and we have to do a second transfer. 01:00: Obviously that s sort of the culmination of the process. I d like to kind of reintroduce Anthony and Denise Steele. Welcome. I know you re looking very pregnant now, so I m sure the process you went through might seem like a blur, but I know that it was a long wait for you. I remember the long wait we had to go through to get ready for all this, but would you just give us a little indication of, either one of you, your journey in getting to this part because you all are the reason we believe so strongly in genetic testing. So would you kind of give us a little history of how you got to this place? 01:00: DENISE STEELE: We have also a five-year-old with sickle cell disease and really wanted to have more children. We tried a second time and I ended up having a miscarriage, so it was very easy for us to make the decision when Niya suffered a stroke at 3Ω years. So for us the decision was very easy to make because we knew we had to do something in order to have another child and not have the stress that we had with the first pregnancy. So far, I can tell you, you have other stresses, but not the stress of whether this child is going to have sickle cell disease or not. So it has been a lot calmer and relaxing pregnancy for me. 01:01: JEFFREY DEATON, MD: Anthony, I m sure being the husband, you have a different perspective on all this. 01:01: ANTHONY STEELE: Yes. It s been quite an interesting journey with the whole process. When you see with Niya s development and the things she s gone through, to do this, we would not think twice about it, to know that we will not have a child that hopefully will not have the disease of sickle cell anemia, and to see that Denise is progressing nicely with this pregnancy, it brings me joy to know that she s going to deliver soon. 01:02: JEFFREY DEATON, MD: That leads to one of our questions, sort of. This is from a viewer. It s for either one of you. Was it worth it to go through all this effort and expense, or if you had to do it all over again, would you just take your chances with another pregnancy? 01:02: DENISE STEELE: I wouldn t take a chance. You know, I did before I found out about this and it ended up in a miscarriage for me, but I wouldn t take a chance. When you live with a child suffering from sickle cell disease or any other chronic illness and you see what they have to go through on a daily basis, as a parent you want the best for your child and I think it s quite easy to make the decision if you have an option to bring in a child. I mean, we still have no guarantees and everything, but if we could avoid sickle cell disease a second time around, to me it s a very simple decision. I mean, nobody wants to see their 3Ω-year-old suffer a stroke or be hospitalized seven times a year. That stuff is not fun to deal with and it takes a toll on your entire family. 01:03: JEFFREY DEATON, MD: How far along are you now?

12 01:03: DENISE STEELE: 23 weeks. 01:03: JEFFREY DEATON, MD: 23 weeks. How do you feel during this pregnancy compared to your previous pregnancy? That s a question from a viewer, by the way. 01:03: DENISE STEELE: I am a lot more relaxed with this pregnancy than I was with Niya. With Niya, there was a lot of nervous energy. I mean, I still pray a lot now, but I prayed really hard then, even harder, but there s a lot of nervous energy and anxiety about the whole thing because you don t know, so from the day that you find out you re pregnant until you deliver, and still when you deliver the baby, you still don t know, so you then have to wait another couple of weeks before, you know, you find out what is going on. You know, it s just really nervewracking. It s nerve-wracking. Especially we didn t know beforehand that Anthony had the sickle cell trait, so that s another thing that I encourage people to do, get tested. If one person has the trait, the other person should automatically get tested. For us it was a little too late when he got tested. 01:04: JEFFREY DEATON, MD: A couple of questions have come in since we ve been talking to the Steeles, but one is for Dr. Koty. I don t think they want you to necessarily list them all, but the question is what single gene defects can you diagnose, maybe an overall number and some examples? 01:04: PATRICK KOTY, PhD: Again, currently you would refer to there s probably over 100 genes that have undergone PGD testing, single gene PGD testing. The main thing is that it has to be a previously identified disease-causing mutation. We can t do it unless it s been already identified and established to be a disease-causing mutation. The other thing is, dependent on what disease it is, again it may have ethical considerations that need to be thought about. 01:05: JEFFREY DEATON, MD: I think we have time for just two more questions. One of them is very fast and I ll address that one, then we have a final question, but another question from a viewer is can you disclose the embryo s sex for non-genetic reasons? That s a great question and it s a very common question we get, but like I said earlier, we have a very good ethics committee at our institution and we did not feel right proceeding with this technology without the backing of the ethics committee at our institution. So they did look at this specific question and we did decide as an institution not to offer genetic testing for sex selection. 01:05: The final question is as follows. I guess this is to myself and maybe Dr. Koty. It s from an IVF lab supervisor. The question is we want to offer this technology to our patients, but we don t have a genetics lab. What are the options? Again, I can start with this one, but Pat, you might need to enter in, if you feel the need to. This genetic testing, as you might imagine, is very high tech and there aren t many places around the country that can offer the single gene testing. We re very fortunate to be one of them, but the nice thing about single gene testing and FISH testing is that the cells taken out can be sent here, tested, and the results can be given back to you, as a practitioner, no matter where you live. So we are able, if you re interested in this technology, Dr. Wininger, our IVF lab director, is very good at training other embryologists to do the biopsy and also training how to fix the tissue in the cells and it s easy to ship it to us overnight. Dr. Koty can do the testing and we can get the report back to you. So if you have an interest in that, just contact us and we can proceed. Anything you want to add to that, Dr. Koty? 01:07: PATRICK KOTY, PhD: Just mainly that, again, if it s for translocation carriers or single gene, we would need the parental blood 6-8 weeks prior to the anticipated starting date so we can get this pre-testing done and then notify the IVF center that it s good to go. 01:07: JEFFREY DEATON, MD: We d be happy to work through all of those details with you all, so if you have an interest in this technology, please let us know. We don t expect every IVF center around the country to duplicate it. 01:07: I believe our time is out and on behalf of myself, Drs. Wininger and Koty, and the Steeles, we know that your time is valuable. It s one of the most valuable things you have and we really appreciate your spending an hour

13 with us. We hope you ve benefited from this webcast. Thank you very much and good night from Wake Forest University Baptist Medical Center. 01:07: NARRATOR: This has been a webcast about recent innovations in reproductive medicine from Wake Forest University Baptist Medical Center in Winston-Salem, North Carolina. Physicians who would like to refer a patient may call Patients who would like to make an appointment or receive more information about the procedure may call