Existing and Emerging Options for Pediatric Oncofertility Patients: Pediatric...

So we start this afternoon with actually an honored guest to join our panel today. Our first speaker today is going to be Doctor Teresa Woodruff. She is the Thomas J. Watkins Professor of Obstetrics and gynecology and the vice chair of research, the chief of the division of reproductive Science in Medicine at Feinberg School of Medicine. Professor of molecular biosciences at the Weinberg College of Arts and Sciences and professor of biomedical engineering in the McCormick School of Engineering, all at Northwestern University. She is also the director of the Center for Reproductive Science, founder and director of the Women's Health Research Institute, and as we know her best, the director of the Onco Fertility Consortium. She's an internationally recognized expert in ovarian biology and then she in fact in 2006 coined the term oncofertility to describe the merging of the two fields of oncology and fertility. She's been a great advocate and supporter of the field of oncofertility and has really helped expand even from the adult model to the pediatric world where we'll be emphasizing today. She'll be joining us from Chicago and we're very excited that she'll be joining us so I will, without further ado, lead into her session which will be existing and emerging options for pediatric oncofertility patients. Thank you, Doctor Woodruff for joining our team for today. Thank you, Doctor Breech. It's really a pleasure to be part of this, uh, global cast today, um, and, uh, to be able to speak a little bit about the, uh, emerging areas, uh, for fertility management, particularly in the pediatric, uh, condition. Doctor Woodruff, could you share your screen with us? All right. Well, so today, we're going to be talking throughout the day about preservation of fertility in the cancer setting. And I think all of us are aware of the uh prodigious rise in the number of cancer uh preserving treatments that include earlier diagnostics, uh, better use of chemotherapy, the emergence of biologic therapy, as well as changes in radiation, particularly in the pediatric cohort, which has resulted in, uh, longevity increases, uh. That are really quite remarkable. From the 70s to today, we have now about 85% of our pediatric cancer patients that are surviving that initial disease. But in the young patients, these same life preserving treatments can in fact threaten fertility, and that's what we want to begin to think about as we go through these first slides as part of this global cast, global cast presentation. So, uh, in the United States, there are, um, 1.4, 1.5 million newly diagnosed cancer patients and about 10 million, uh, global cases of cancer. And if you look at the demographics for those who are in the younger age range, about 10% are in. The age of 45 and younger, and about 11% of our breast cancer patients are diagnosed before the age of 40. So, really, uh, about 200,000 individuals each year who are in this younger age range of post, uh, uh, uh, postpubertal uh age range. One of the easiest ways to uh see the uh issues associated with cancer therapy is shown on this graph from Paula Goodwin, and that is the probability of early menopause in the breast cancer population. If you look at the estimated probability of entering uh uh menopause uh for um the healthy population in the red line, those individuals with no treatment and no cancer, they reach, uh, uh, menopause. Around 51.5 years of age. But with the addition of either hormone therapy, chemotherapy, or in fact, both, you see that those patients enter into menopause at significantly earlier ages than the uh control population. Uh, and this represents uh the population that is at risk for losing not only fertility, but the endocrine support that the ovarian tissue provides to help the, uh, bone and other tissues in the periphery. Um, we started with the adult population, but even as we were developing strategies that were avail that could be made available to the adult population, pediatric oncofertility began to emerge as a really urgently unmet need, again, primarily because of the success in treating uh young cancer patients and their ability to survive that initial disease. Um, but, um, these same cancer patients will be significantly more likely to be infertile, um, than their, uh, siblings. And so this particular issue, um, has largely come to the fore in the last decade, and for which we'll be describing today some of the options that are available for our pediatric cohort. Uh, and even as we were extending the age range from the adult population down to the pediatric population, uh, the fertility concerns even beyond cancer treatment began to be, uh, recognized by, uh, members of the oncofertility community. And these included young patients who present with rheumatologic disease, a disease that largely presents in female, uh, and for whom those treatments are cytotoxic, uh, and, uh, can in fact interrupt a normal gonadal function or can be counterindicated for, uh, pregnancy. Uh, also, um, patients who have, uh, beta thalassemia, particularly those patients who will receive a stem cell transplant. Uh, this can be associated with, uh, uh, sterilizing whole body radiation that, uh, many patients are unaware, uh, can in fact, um, uh, be, uh, fertility, uh, limiting. We also have a number of individuals who are having earlier and earlier diagnostics for genetic mutations that lead to a loss of fertility and early menopause. And this would include Turner syndrome, and particularly in the pediatric condition, there are a number of parents of uh patients with Turner syndrome. And here we're Largely talking about those children with the mosaic disease that in fact will um enter into an early, uh, a premature ovarian failure and for whom fertility management is something that um many physicians and parents uh are very interested and anxious in, in managing. And then there are those individuals who carry a mutation that predisposes them to an early risk for cancer. Uh, these are often diagnosed, uh, after 18 years of age, primarily because our genetic counselors are not looking at genetic, uh, diagnoses earlier than that. Um, but those represent another category of individuals for whom fertility concerns, uh, may, uh, become, uh, important and will come in uh to your practice. And then finally, the group that is probably the largest uh growth area of physicians with uh interest in managing the fertility and endocrine issues uh associated with those patients who have disorders of sex development or in fact our pediatric cases where transgender and transitioning between sexes. Uh, uh, in both of these cases, the, um, biological gamete, um, may, uh, be, uh, at risk as the individual is transitioning between, uh, between genders. And so there may be both parental as well as, uh, the individual's interest in maintaining, uh, the health of that biological gamete for later, uh, fertility. So, as we've thought through this problem, uh, really, fertility preservation was an option for men for many, many years. And even for pubertal boys, uh, and those pubertal boys and adult men could in fact, um, provide a sperm, uh, semen sample, uh, at the time or prior to the first, uh, sterilizing treatment, uh, and that would allow for the management of their long-term fertility risk. Uh, even though the, the, uh, ability to bank semen is relatively easy, but the cost is relatively low, navigation wasn't always provided for our male patients, and that's something that, um, we all as a community have worked to, uh, provide better options, uh, better navigation for those individuals. However, young women with the same hope for survival of their initial diagnosis have had traditionally fewer options made available to them. And this is really because of three main gaps. One is the information gap, and that information gap is really that our providers didn't know that these young women who have been treated for their disease and then uh survived, um. That, um, that, uh, that disease and the treatment were actually going through profound, uh, infertility or uh sterility. And so now that we have a large number of patients who have survived the disease, we know that this is a major issue. So it's critically important that uh oncologists really at that earliest time of diagnosis, talk with the patient about this issue. We also had a data gap. We didn't know exactly which patient um would in fact be sterilized and which might just have infertility with some amount of fertility uh main uh being maintained. And I would say this is still an issue for the field that we don't always know on an individual or personalized level, uh, who's going to, uh, recover, um, some amount of gonado function even with the most profound treatment and who will actually have uh no fertility remaining. And then, uh, the last issue is the option gap. And the option gap is that at the beginning, we really didn't have options for even young adult cancer patients, let alone pediatric cancer patients. And now, as you'll see today, we're filling in that option gap with existing, uh, assisted reproductive, uh, interventions as well as with, uh, the experimental options that I'll bring you up to date on today. And so those gaps are ones that we're, that we are working on, and I think really. Really the one that we've been the most successful on is making sure that interdisciplinary teams such as those at the University of Cincinnati uh are able to work together across fields and be able to provide uh the best information to parents and children about their fertility risks in the cancer setting. So we've done this through an interdisciplinary framework which said that we in fact had to invest in uh some of the fundamental questions about ovarian follicle development uh and how we could protect eggs and the ovarian tissue for long term without damage. This was something that 10 years ago, we really hadn't addressed, uh, and certainly semen samples have been maintained for decades without appreciable damage. Um, but really being able to, uh, mature follicles in vitro as well as protect them for a patient who might be 5 years old at the time of diagnosis, and that tissue needs to be cryo-preserved for 2 or 3 decades before it would be used is something that we had to deal with. That requires a scientific community to come together around new ideas and new approaches, and you'll hear about some of those today. I already mentioned that the information gap uh between the um patients and the provider, and indeed between providers themselves, where we really needed to bring individuals together who formally didn't talk to each other, where reproductive endocrinologists and oncologists and pediatrics really weren't in the same uh dimension. And, uh, not only did we need to create a, a common language that allowed for, uh, these folks to be able to To communicate, we also needed to create silo spanners, and those silo spanners include the patient navigators that you'll be hearing about, uh, hearing from later today. They represent individuals who are able to effectively go between disciplines and sometimes reach across very disparate disciplines, uh, between radiation oncology, surgical oncology, uh, between plastics and genetics and reproductive intervention, all of while, uh, maintaining, uh, the patients. Um, need, uh, to, um, survive that disease is paramount, uh, as they're being navigated through all of these different intricacies of the, uh, medical environment. And we wanted to bring new scholars, uh, into this field. At the beginning, we really didn't have a common language, uh, we were really siloed, and the patients were not getting the kind of treatments that they are today. And part of the success of what you'll see today, again from the University of Cincinnati's, uh, group that is organizing this. is that we have a whole group of new scholars who have come up through the medical disciplines, understanding how to think in an interdisciplinary way. This required a number of hospital fertility multidisciplinary patient management boards, and you'll hear more today about how to develop those in your own setting. As we were developing both the basic science and this uh medical management strategy, we also had patients whose urgent unmet needs needed to be met today. Even when we didn't have all the answers and weren't perfectly prismatic on the way every treatment would actually affect an individual, we needed to have options made available both to the cancer survivor and to the parents in whose interests we work. And so that also required us to do 360 thinking to make sure we weren't just figuring out basic Science approaches that would end up in a paper, but that we could really develop the medical management strategies for those individuals, giving them the options that were available to them at the, at the time uh that they came to the program. And that has changed dramatically over time and the field continues to change. And that's one of the things that I think uh you'll hear more about today, uh, and if you come to the ACA fertility meeting in November, is that each year there are real advances that are made that move into clinical. care that weren't there last year, uh, and that's really important to know. But on the patient side we've also developed strategies for legal issues, ethical issues, insurance and reimbursement, and knowing how to talk to patients about religious concerns they might have. So that's really the 360 of care that, uh, I'll talk about. And so this has led to then the Onco Fertility Consortium. You'll note there's no hyphen between onCO and fertility, uh, and that is because sometimes our oncologists really don't see fertility as something that is their issue. Um, uh, and we wanna make sure that this isn't just a simple fertility consult, but rather that oncofertility really represents all of the dimensions of what a young cancer patient is facing and how they will experience fertility care on their way back to, uh, a healthy, uh, life. So, we do have a National Physicians cooperative that is an infrastructure for partnering between science and medicine to enable the most rapid communication of information uh from the bench to the bedside, and in turn, uh, hear from the uh bedside to know what it is we need to focus on next. And not only are there local, uh, there, uh, national sites, but there are global sites that are all linked together. Of, uh, they're also, in addition to the 71 adult sites, there are 19 pediatric sites, uh, that are part of the consortium. So now, let me quickly take you through some of the options that exist for patients today. Uh, and, um, I first want to note that since 2010, uh, our major, um, guide, major, uh, um, clinical um societies have developed practice guidelines. And this is really critical for individuals who are on this global cast who are new to this field. It is that the clinic of the ASCO-American Society of Clinical Oncology, as well as the American Society for Reproductive Medicine, and our Pediatric Hematology, Oncology, and Nursing Society, among many other societies, all have strong guidance to physicians that says largely the same thing. So first, really make sure that you're talking at the earliest possible moment of About a fertility impairment associated with either the disease or the treatment and refer that patient to a qualified aqua fertility specialist as quickly as possible. And thirdly, to continue to promote the research that's gonna be necessary to advance the state of knowledge so that tomorrow's patient is not treated the same as today, which after all is the promise of basic science in medicine. The American Academy of Pediatrics has had additional guidance which has been very important, uh, within the field of pediatric and adolescent uh oncofertility, and that is that the parents may in fact act to preserve fertility of cancer patients, um, uh, and that has really been an important part of the evolution of. This field because at the very beginning, it was not clear whether or not one could intervene on behalf of the fertility of that future uh individual because the fertility was not impaired at the time of the uh diagnosis, but AAP has uh has uh provided guidance on this topic. Now the options for women are listed here, and they include, um, embryo or egg banking, and that is for women who are 18 and above in the Onco Fertility Consortium we decided 2 years ago as a consortium to move the uh. Age boundaries downward uh for uh for egg banking from 18 to 16 uh but below that um that is not part of the onco fertility guidance um there are individuals who are doing it below that age and that definitely should be done under IRB approval only. Um, for some individuals, uh, they will not be able to go through either mature or emerging technologies, and so adoption may be an option for the future of that individual. The Oncote Consortium has a list of 12 adoptive services, uh, services that are, uh, cancer, uh, friendly. Not all patients will have the ability to bear their own pregnancy. So, uh, that treatment, either radiation or chemotherapy, may permit some amount of uh normal ovarian function, uh, but that uterus may. No longer be capable of implantation. So surrogacy may be, uh, may be a necessary part of the future option for that individual. We also want to make sure that even as we're talking about fertility and fertility loss, that we're also thinking about endocrine health. And, uh, not all patients will be sterilized immediately by the cancer treatment. They may go through a period of, uh, of, uh, fertility where they are going through normal cycles before they go into an early are fertile, they may not be aware that they could in fact have a natural pregnancy. And if that's intended, that's really an important option for that cancer patient. But we also want to know that, uh, we want to appreciate that some cancer patients will think that they are going to be sterilized by the treatment and won't realize that they actually can have a conception, uh, if their ovary uh is functional. And so that means that we need to talk to young cancer patients about sexuality and contraception, that even in the cancer survivor condition, that, um, contraception is important to avoid unintended pregnancies. Uh, and, uh, for some physicians, particularly oncologists who are quite wary of, uh, endocrine hormones and their ability to exacerbate cancers, even those cancers for which there is no, uh, evidence of hormonal interplay, um, we wanna make sure that Those oncologists appreciate that there are a variety of different uh contraceptive options, not just hormonal, but also, um, barrier methods. And cancer patients should be advised of this. And pediatric cancer patients as they transition through puberty should be advised of this. So, um, today for adult cancer patients, uh, the time for waiting, uh, for mature technologies currently is a 12-day average wait at throughout our onco fertility Consortium site, uh, and there are options available, uh, for fertility management even in the hormone positive setting. And for those breast cancer patients, uh, tamoxifen holidays allow the patient to become pregnant with some, with good outcomes. And so this will allow patients to be, to be more compliant to their long-term uh hormonal or chemotherapy, uh, which is going to be important for their long-term survival. For some patients who don't have the option for um for uh hormonal intervention which includes uh all our pediatric and adolescent patients at least up to the age of 16, um, their options are really limited to ovarian tissue cryopreservation and ovarian tissue cryopreservation, uh, requires the or uh permits the removal. Under IRB approved consent at each individual site, uh, either an entire ovary or a biopsy of the ovary. Uh, and that ovary is then prepared into small cortical pieces, which is what you see in panel D. That's the outer cortex of a patient, uh, with small follicles, as you can see in panel E. Um, from the small follicles all The way to the large antral follicles that are maintained in that tissue. That tissue has been cryo-preserved for that patient's later use. And as I mentioned, if you're talking about a 5 or 6 year old, that tissue may be cryo cry cryopreserved for 20 or 30 years. In the top panel are those oocytes that come out adventitiously when you're dissecting that tissue. It's important even for pediatric cases to remove that Ocyte. And it depends on the site whether or not that Ocyte is matured and stored for the patient's later use, or whether or not it goes into the research portfolio. In many settings, it is for research because in fact, we don't know the quality of the Ocyte in the patients 16 years and younger. And in fact, In the adult annuploidy literature, we know that annuploidy rates increase dramatically after 35 years of age, but we also know as you go to younger ages as the annuploidy rate decreases, it then again increases. As you go below 18 years of age, and we've repeated that data with uh IBM uh acquired O sites from the National Physicians Cooperative. So pediatric, uh, pediatric, uh, oncofertility sites are well advised to consider picking up those O sites and uh collaborating with researchers within the on fertility consortium so that we can determine what the quality of those gametes actually might be. Many members of the Global cast community may be surprised to know that from that ovarian tissue that we've cryopreserved, um, prior to the treatment for these cancer patients have been now documented 86 live human births. And these have occurred in the United States and around the globe, and that really is good news because that says that the way we're cryopreserving this tissue maintains its function. The issue for us is that we don't know the efficacy or the cancer recurrence for cancer patients. The efficiency says we don't know globally what the The denominator is for those transplants. And the cancer recurrence is that in many of the cases where ovarian tissue is removed, cancer still is residing within that tissue. So that requires us to continue to do the research that we hope is going to make that tissue useful for every patient under your care in the future. And this is particularly important for our pediatric cancer patients. So, um, as you know, the follicles within the ovary, uh, are present at the time of birth, uh, being formed between the 2nd and 3rd trimester. And so an ovarian cortical tissue from a pediatric cancer patient is actually more well endowed with potential, uh, fertility than the adult ovary. You can see that in the bottom right-hand corner is a slice of ovarian cortical tissue. From one of our NPC, our National Physicians Cooperative site, from a patient who was 4 years old, and you can see embedded in that tissue a large number of dormant primordial follicles. What you can also see in that same tissue, if you actually stain it for the cancer that that patient had, is a large number of circulating cancer cells. So if that patient tissue were transplanted back into that, into that adult survivor, that 4-year-old, uh, now let's say is 34 years old. That may reintroduce the cancer that that patient just survived. So for our cancer patients, we have to really think about fertility and endocrine needs, and I'd like to bring you up to date on some of the ways we're trying to address it through follicle maturation and the development of tests for high fidelity oocyte maturation, through the development of strategies for endocrine hormone production in order to enable pubertal transition because many of our prepubertal patients are unable to go through unaided, uh, puberty. And that will hopefully lead to cyclical hormones that supports overall systemic health for those individuals. So for those of you who are not used to looking at ovaries, uh, this on the, on, on, uh, your left, I think is the, uh, ovary of a rodent. And throughout this talk, I'll be going back and forth, uh, through between uh rodent and human tissue. I'll try to be very careful so you know where we are with the development of these strategies when we've reached human and when we're not there yet. So the The ovary contains those large follicles, and if you cut down through one of the rodent follicles, what you'll see in the middle is the oocyte nestled in a set of cells called cumulus cells, and the surrounding cells are the endocrine cells that are producing estrogen and progesterone. That follicle is growing and developing in natural space. It's moving to the outside of the ovary in the bottom right hand corner and Soon that ovary will reach the outer space and under the uh hormonal control will break down the outer surface and ovulation. The egg will move from the inside to the outside, and if sperm is available, it's shown in the primate embryo on the right, it will fertilize that egg and you will have the development of the embryo. So our strategy is to try and grow those follicles to get high quality eggs, uh, that can be fertilized. But if you take that ovarian tissue and put it on flat plastic, you can assemble follicle-like structures. But in fact, the egg that sits interior does not mature at a high rate. And more importantly, when you take human tissue and culture in the same way, you don't get that, uh, follicle-like structure. The oocyte simply sits flattened out, uh, on the plastic. So we ask whether or not the structural context of that follicle actually matters to the developmental competence. In other words, if we can maintain that three-dimensional architecture, could we mature that egg? So together with Lonnie Shea, who's a biomedical engineer, we took the ovarian tissue and the top panel shows from uh one of the pediatric patients again, and you can section through and actually optically see those small follicles. We can then isolate the follicles of various stages and shown on the bottom are follicles from the human on the left and the mouse on the right. The follicles can then be placed in a biomaterial called alginate, and those follicles then can be matured in vitro. And our goal is to mature the follicle, uh, both hormonal function as well as, uh, the gametes. You can see that the follicles begin to expand in the top left panel, you can see, uh, many more granulosa cells, and uh they differentiate into fecas cells in the bottom panel. This means that they have the ability to make both testosterone and, and progesterone, and then convert that into estrogen, which is shown on the right-hand panel, where the follicles at the beginning make no appreciable hormones, but they awaken over time in our culture. You're now looking at a three-dimensional follicle with the antrum in the center, and the oocyte has now moved to one side with its cumulus cells. And when we stimulate this follicle in vitro, you can achieve an in vitro ovulation, where a rupture on one side of the tissue is developed and the ocyte, a mature oocyte, moves from the inside to the outside with its surrounding cumulus cells. This is a whole pan. Panel of follicles where you can see that each one of these follicles is going through this very specific physiology that you would have to see in that you would expect to see if the follicle was going through all the stages of maturation necessary to get a, a, a high quality gamete. And we can further prove that because we can take these high-quality gametes, we can fertilize them, and we have live healthy births. So, um, given that we have hormonal production, we asked whether or not we could support ovarian follicle uh hormone production over, uh, the entire, uh, time sequence of a menstrual cycle. And the goal here was to determine if our in vitro cultures were supporting not just the early stages of folliculogenesis, but all of the stages of folliculogenesis. And if it were not, could we engineer a way to better enable follicle development? Because if So, that would be immediately transferable to the, to the human. So we took advantage of a microfluidic system that is shown here uh that allows us to connect individual tissues or multiple tissues together in a single circuit. And for today's conversation, it's not necessary to go through all these details, but simply put, we have a series of systems that allows us to interrogate these isolated follicles from the tissues in a new and robust way. This was done in collaboration with an engineering group at Draper Laboratories, and Xuo Shuo, shown in the front, was the one who was in the laboratory managing all these uh various systems. And I'll only show you one system today, which is what we call the Evatar, and this includes uh a mouse ovary together with human fallopian tube, human uterus, human cervix, and Human liver for metabolism and our ultimate goal, as I said, is to look at the endocrine function of these tissues together, but we also believe that eventually this will be personalized so that we'll be able to look at drug interactions on an individualized basis to see whether or not there will be toxicity or efficacy on a particular individual for a particular drug. And you can see just again, one piece of data from this exciting new technology that as you look across the 28 day cycle, we're able to engineer a full follicular phase with the rise and fall of estradiol prompted by HCG at mid-cycle and the rise of progesterone for a full luteal phase. We also have the downstream tissue, so fallopian tube functioning can be maintained for the full 28 days, and we can actually add in the hormones of pregnancy and maintain progesterone for an entire luteal phase. So this suggests to all of you who are listening that the way we're engineering these follicles is in fact phenocopying what we see in vivo. And that was something very important because we are not going to be able to Transfer that egg to uh a patient where if if there is a patient who's ready to go we will attempt that, but we can't do that on the research side. So our goal is to try and show as well as possible that these follicles and the associated eggs are of high quality. So I've just shown you the work in the mouse and what you're seeing on the bottom panel are human ovarian follicles grown for 30 days and in fact we've been able to get these follicles to mature. Uh, after 40 days, uh, and, um, I'll simply show you a few of these follicles. This is one of the human follicles that's been growing for a few days. You can see the O site is now moving to one side. Uh, at the end of 40 days we can stimulate the follicle and can recover GV intact eggs that can be matured. And for the first time 2 years ago in 2015, we pre-reported for the first time ever human, uh, human eggs, human. Into eggs that have been matured completely in vitro, so this is important for the clinicians to know because this is one of the new technologies that's being developed that we hope ultimately will take advantage of that tissue that the patients have uh been banking for later use but for whom that tissue also has has carries with it a significant risk for reintroducing the disease so it would not be a good candidate for tissue transplant. We also want to know if that tissue is of high quality and so we're developing new mechanisms for being able to measure uh egg quality and one of those is by the zinc spark. This is in fact a uh a metric that we hope will actually allow us to see the release of zinc from an individual oocyte as you can see in that little corona there, and uh the egg will release that at the time of fertilization. If the egg is not. Not able to be matured, then that egg cannot be fertilized. We have now translated this to the human. What you're seeing here are human eggs. The one on the left is not mature. The two on the right are mature, and in just a moment you're going to be seeing the release of zinc from those 20 sites. So that would be a good signature for us that in fact the egg is of high quality. We're not able to do the zinc, uh, spark on the, uh, tissue from the. NPC at this point because that's funded by the National Institutes of Health, but if we're able to get non-federal funding, we'll be able to examine whether or not the follicles that are grown in vitro from the pediatric cancer patients actually support oocyte maturation through parthenogenic activation and give us good, uh, zinc sparks. If we can show that we think that then those eggs are going to be ready for that patient's, uh, later use. This is just one, movie. I don't know if we can show that, but this shows an individual sperm at the time of entry into the O site. It's the kind of uh image that you just rarely get, so I'm gonna try and show it to you. You should look at this sperm right here. It's actually in the field. It's about to go through the outer surface of the egg. It's just popped through and in just a moment you saw the thing spark. So this gives us a very good information that that egg is of high quality. So what makes a good egg? Of course, we have to have good chromosome structure and number. Uh, we have to have the right genes which we can do by polar body analysis or by blastocysts, uh, by PGD, and we can also look at zinc as an extracellular regulator, uh, measure of egg quality. So for these pediatric cancer patients, I've told you that now we can grow the follicles and we have eggs uh as far as we can go under the authority of NIH. But we also wanted to develop a scaffold that could restore puberty to the young cancer patients. So, Monica Laonda, who was in my laboratory as a postdoctoral fellow, and now a faculty member at Eury Children's Hospital, took on this project of uh removing the cells from the ovarian tissue. These are from pediatric cancer patients. And with that decellulized tissue, which you can see here, the skeleton of an internal organ, the skeleton of an ovary, she was able to uh replace the cells onto a bio. punch of that tissue and that recellularized that graft and then transplant it back into a prepubertal overacting mice mouth and 100% of the mice with these transplants were able to go through puberty. So that was really important news, but of course that scaffold is not going to be the kind of scaffold that we use uh for the long term. So we've developed a number of scaffolds including taking that uh bio. The biomaterial from the ovary and making it into what we call an ovary paper. Uh, this will be published next week in advanced materials, and we can take that tissue and put a human follicle, as you see in the bottom left, onto that tissue, and we can then suture it down to soft tissues like the region where an ovary might have been removed for a young cancer patient or where an ovary exists but it's no longer functional. So this allows us to. Functionalize those follicles and essentially reduce the number of cancer cells that might be transferred with that with that tissue. We also can take that material and 3D print it, and we've been able to 3D print using at this point gelatin published with gelatin, and we can make a bio-inspired scaffold that looks much like the ovary and then place into it, uh, ovarian follicles from prepubertal animals and those prepuber. ovaries are GFP labeled, uh, so that we can see the difference between the follicles that are transplanted and the host, um, mother. Uh, we're able to see that vascularity comes into the bioprosthetic, which I think is the most exciting part of this, uh, new soft tissue transplant. And we have live offspring, and again, since they are GFP labeled, you can see that we have, uh, live pups. So that means that this This is first soft organ transplant with fully 3D printed bio-inspired tissues, uh, is enabled within the mouse. Uh, we are now moving on to, uh, do the same kind of uh steps in the mini pig, and we'll be taking some of the scaffolds and try to transplant them into, uh, uh, humans who are consenting for recipients of a small piece of the bile, uh, bile, um, scaffold under the arm. So what I've been able to do is share with you a little bit about what we're doing, uh, in terms of the new technology that will eventually come primarily for the pediatric cancer patients. This is for follicle maturation with we hope high fidelity oocyte maturation with tissues that can support endocrine production that will enable a normal, not only pubertal transition, but also ongoing cyclical hormone that will support uh systemic health. We've told, talked today very globally about the fact that we do have a global oncofertility community, and the University of Cincinnati has been a tremendous leader in the area of oncofertility, and they are there to answer questions that any of you might have about this field. I've also told you that we have human M2 eggs from encapsulated in vitro follicle growth. This is the first time human M2 eggs have been matured entirely in vitro, and that's very exciting for our field. I've also Shown you new discoveries about the fact that the human egg releases zinc as a marker of health, and we hope to be able to apply that to the pediatric uh tissues in the future. I've also shown you the menstrual cycle in a dish with a variety of tissues linked together by the hormones that can cycle over a 28 day time period and how we've translated that into ovarian, uh, bioprosthetics, and that bioprosthetic led to live births in mice. So we hope in the future that we're not just uh publishing papers, but we're actually making progress towards the patient's needs. And we think that by the time we get to 2027, 10 years from now, as oncologists, you will have better cancer control and treatment strategies that hopefully will reduce the number of cancer patients for whom off-target effects represents a major concern. Um, we also believe that there will be neoadjuvant fertileprotective therapy. Doctor Soo Yung Kim, who's in my laboratory, uh, at this point, a research assistant professor, uh, is developing these next generation neoadjuvant fertilerotectives that I think will represent, uh, the needed urgently needed medical management for cancer patients. If we could not take out. An ovary from a cancer patient in the future that would in fact be our goal and so that is what she's developing actively. Uh, I think we will have in vitro follicle maturation and some center will collaborate with us to look at the oocyte maturation and as I said, um, we're developing these new ovarian bioprosthetics on our way to hopefully eliminating the field because cancer, uh, treatment has gotten much better. So with that, uh, I hope what I've done is told you that um we have a community of practice that links basic science to medicine. And when these grants and papers meet a clinical problem, patient needs are met and can change what really is a, uh, originally a devastating diagnosis into a series of life-affirming interventions and what we now like to think of as bench to bedside to babies. So with that, I'd like to thank all of you for participating in this global cast, uh, to thank the University of Cincinnati for organizing this event, and I'm happy to take any questions if time remains. Thank you. Doctor Woodruff, that was a wonderful presentation, not only informative but interesting and encouraging about the future care of all of our patients. Um, we have enjoyed working with you, uh, here from the Cincinnati Children's Hospital Medical Center as well as our relationship with the University of Cincinnati. I was wondering if I might pose a question to you as well, having been an innovator in the field, uh, sharing your passion for the care of patients. I think that many who have signed on to the session today are probably just beginning their programs, and you being the leader in the field of onco fertility were really the initiator of the entire field. Would you have any advice for those who might be logging on today and really just starting their program where they are about how they can really start the baby steps to move forward for an outstanding program over time? Well, I thank you for that question. I do think that when you start out, it may seem daunting, and the first thing that I think is important is that there now are groups that have gone down this path before you. And so, for example, I would reach out immediately to Leslie, uh, to you, Leslie, and ask you how you've put together your program, and I think you'll be talking about that throughout this afternoon session. Um, so there are people who you can be connected with throughout the National Physicians Cooperative, including, uh, members of the 19 centers, uh, that, uh, focus on pediatric oncofertility. So you can, uh, email me or email the program or email Doctor Breach, and we can immediately, uh, connect you with, um, the folks who will, uh, identify. By people in your vicinity who already have a program who you can actually uh template from that you also can find all of the resources for how to start a program uh at the website that's there, the aquafertility.northwestern.edu. Uh, there are a series of materials that are provided there including the IRBs that you would need in order to do some of the. The, uh, experimental, um, tissue recovery, um, and, uh, so all of that can be found within that site. The last thing I would say is that the pediatric network of the Aqua Consortium is extraordinarily, um, well organized. They have quarterly meetings and in fact this last year we published a book that has almost everything you need to know at a very up to-date. Um, at a very, uh, contemporary way of the issues associated with pediatric oncofertility, and that book is, um, by Springer. You can find it on the website, but I think it's one of the most essential books that tells you not only how to set up a program, but what some of the options are and what some of the boundary conditions are. So I would urge you to pull that resource as quickly as possible. Uh, excellent advice as always. There actually is a question that we have from one of the participants. I was going to forward that along to you. Um, the, the participant says, so you have successfully had 44 patient tissue produce 65 follicles. Have those follicles been able to produce eggs that have been able to be successfully cryopreserved? So those follicles, so, um, there were 6, there were 44 patients that were recruited over a one-year period of time from the National Physicians Cooperative uh uh tissues that came in for a specific period of time, all tissues went into this one protocol. And, um, from those tissues, and that, uh, was largely very small biopsy tissues, we were able to isolate 44 follicles. Those were all fresh tissues. Not frozen tissues. The vast majority of those tissues were actually um uh transported. They were transported fresh throughout the network. Um, and then from those 44 follicles, um, some number actually uh did uh mature. I think it was half of those follicles. I'm forgetting the statistics. Um, I think it was something like 20 of those follicles actually went on to make an Antrim, and from those we got 44 M2 eggs. We got the remainder were GV eggs. And so it was uh 4 out of 4 out of 20 that actually the follicle uh mechanics grew, um, so you know that is actually both good and heartening, uh, and that also represents um, the where we need to move from we need to develop additional strategies for being able to grow those follicles. Uh, part of it might have been aided by actually cry preserving the tissues at the site. And then, uh, thawing the cryo-preserved tissues and growing them because there was some damage to the tissue by, uh, just transporting for extended periods of time. Um, but in this particular case, they were all fresh tissues that were being, uh, utilized in that particular study. It's, uh, reported in Scientific Reports 2015. Excellent so I'll just, um, if you have a moment, um, because we have you for a short period of the global cast and not here for the entire afternoon, there was a question also from a participant really about sort of criteria for determining appropriateness for ovarian tissue cryopreservation. Obviously this is focused more on the pediatric population and so that question arises we will be addressing some of that as we continue on the panel this afternoon but knowing that you won't be joining us for the entire afternoon just wanted to throw that one at you to get you a little bit of a comment. Your side as well. Well, you know, so I am the PhD and I rely on the MDs for knowing which patients to actually, uh, take through the ovarian tissue cryopreservation protocol, but I'm very heartened that we just did an analysis, and this was done by Doctor Francesca Duncan of the NPC tissues in the pediatric from 0 for 1 month to 8 to 15 years of age. Uh, and, uh, to look at the ICDN 10 numbers to see if we were actually, if the clinicians throughout the network were in fact identifying the either the, uh, disease or the treatment that was most likely to have a sterilizing effect. Uh, in other words, um, were the, were the physicians throughout the network making good choices. And the answer is yes. And this was presented at last year's Onco Fertility meeting, uh, and, um, uh, that will be published, we hope very shortly. And so for anyone who is interested in getting started in this field, I guess what I would say is you are among the experts. And so people like Doctor Breach and those throughout the pediatric network are all using uh criteria that allow them to identify those patients most at risk. And really there we're talking about the females. Again for the males, it won't matter whether they're at risk or not. Uh, if you have a Mother who is very concerned about fertility, even if it isn't a high risk case, uh, they can go ahead and bank semen because it's a relatively easy non-operative, uh, option. It simply costs money and maybe 6 hours of time for the female case that, uh, is not the option there. It is an intervention. It's a surgical removal of. Part or an entire ovary. Uh, and so it's really important that all the oncologists actually, uh, have a strategies for identifying who would be uh most likely to, um, to require this kind of intervention. And I think it's changed over time. At the very beginning of the consortium, of course, there were no pediatric cases. It was all adults, and in fact, it was all adult breast cancer. And of course, today we have nearly no adults that are part of the National Physicians Cooperative Tissue Cryp Preservation Program because those patients have other options. And so, um, this is a field that continually moves and where the clinicians are continued to track those patients long term to try and understand what the fertility issues are, uh, and then, uh, I really can't emphasize enough the value of convening. Uh, and so every year in November, we convene in Chicago, and the purpose is to really touch bases with each other and find out what people now know. And in many cases, to design the experiments that are gonna be necessary to develop new strate. Strategies for the future. And in the absence of having that time to convene, science would emerge and medicine would emerge as it always does, very slowly by papers getting published and maybe a year and a half since the time that you knew something, it would finally get into the literature. And what we've done to the consortium is to say that that's too slow for what it is that we want to accomplish on behalf of the patients. So we all come together in the Aqua fertility Consortium, and clinicians take time off, which is really remarkable, uh, patient navigators, nurse navigators, um, uh, psychologists, um, trainees, your Neurologists, surgery, we all come together and the idea is to share as much information as we can and in so doing on an annual basis, we make the field better. And so I think in answer to that question, there's gonna be a lot of probably detailed conversation about uh each of the cancer types and treatments that you'll show in tab table form later, but I guess my point is that we can. And be able to uh uh do best on behalf of the patients. In in this time where it is what based on what we know now. Well again you've been awesome as usual and I think that we are excited about being able to continue the conversation today you've alluded to there are a lot of individualized decisions and criteria that we can discuss, but it's that continued dialog that's critical to moving the field forward. And we appreciate you starting the dialogue today on a fabulous note and we will continue our discussion this afternoon. Thank you so much for your time today and your continued support of the field and our program as well. We hope you have a great afternoon and we'll continue our discussion here in Cincinnati. Thank you so much.