Well, welcome to the final plenary of ENDO 2022. This is going to focus on the challenges of fertility and reproduction. And we have two excellent speakers ready to delve into this important topic. But first, we're recognizing our final two Laureate Award recipients. And just a reminder, the award nominations for 2024 are currently open. So I highly encourage each and every one of you to look through the list and to nominate one or more of your colleagues for awards for 2024. So our first award is the Outstanding Public Service Award. Established in 2013, it is presented to an individual who best demonstrates dedication to public awareness or public service in support of the field of endocrinology and the patients who suffer from endocrine disorders. This year's Outstanding Public Service Award recipient is Dr. Rita Cugliani, an Associate Professor of Medicine in the Division of Endocrinology, Diabetes, and Metabolism at Johns Hopkins University School of Medicine in Baltimore, Maryland. An active clinician who sees patients regularly in the Johns Hopkins Comprehensive Diabetes Center, Dr. Cugliani directs the Diabetes Management Service for Johns Hopkins Total Pancreatectomy Islet Auto Transplant Program. She has served on the Board of Directors for Diabetes Sisters, a national organization working to improve the quality of life of women with diabetes. Dr. Cugliani has participated in Endocrine Society Hill Days and has served as a society spokesman on insulin affordability. She currently sits on several committees, including the Clinical Practice Guideline Task Force and the EndoCares Steering Team. I want to thank nominator Rob Lash with letters of support from Des Schatz and Paul Ladenson. And congratulations to Dr. Cugliani, an outstanding clinician, educator, and research who has dedicated her talents and energy to broader public awareness of public health initiatives in endocrinology, diabetes, and metabolism. Let's welcome Dr. Cugliani to the stage. Thanks so much, Carol, and thanks to the Endocrine Society for this honor. I would specifically like to thank Rob Lash and my nominators also for this recognition. As endocrinologists, we care for people with diseases that are exceptionally common to those that are extremely rare. However, our responsibility to our patients does not end once they leave the clinic. The communities in which our patients live has a tremendous influence on their health. Further, while the discovery of knowledge is clearly important, the responsible dissemination of that knowledge to people most in need is of critical importance to improve human health. It has been a privilege to lead population-based initiatives, to lead diabetes care in the communities, and to develop rigorous clinical practice guidelines based on the latest evidence. Also the opportunity to advocate for insulin affordability and for increased research funding as part of Endocrine Society Health Days is unparalleled. Last but not least, I would like to thank my family who could not be here today, my husband, Sachin, and my children, Sean and Sonia, for their unwavering support. Thank you so much for this recognition. Congratulations. Our final award for this year is the Sidney H. Engbar Distinguished Service Award, which recognizes distinguished service to the Endocrine Society and the field of endocrinology. This year, the Endocrine Society is pleased to recognize Dr. Simon Rhodes, Provost and Vice President for Academic Affairs at the University of North Florida in Jacksonville. For over 15 years, Dr. Rhodes has reached trainees and early career investigators around the world and has given special attention to diversity, equity, and inclusion. He has been engaged in the FLARE program since its inception and has served as a program presenter and mentor for over eight years. Dr. Rhodes was also co-chair of the Endocrine Society's Trainee and Career Development Core Committee and a member of the Minorities Affair Committee. He currently serves on the Society's Finance and Audit Committee and the Global Leadership Academy Planning and Advisory Task Force. Thank you to nominator Sally Kamper with letters of support from Lauren Fischbein and Kristen Vella. Congratulations to Dr. Simon Rhodes, who has made a difference in diversity, equity, and inclusion for the Endocrine Society and has served the Society faithfully and effectively in inspiring the next generation of endocrine scientists. Please help me in welcoming Dr. Rhodes to the stage. Thank you, Dr. Wysham. I'd first like to thank my wonderful wife, Jan, and our kids, Samantha and Mitchell. I thank the Society, Dr. Ross, and the Committee. I thank Dr. Sally Kamper. She has been a mentor, a friend, and a role model for me. I thank our great Society staff. I thank all the colleagues, collaborators, and committee members that I have worked with, especially the Friday Morning Group and the Flair folks. I thank my amazing students and fellows. I thank Dr. Tom Landefield, who first encouraged me into Society service many years ago with the formerly Minority Affairs Committee. I encourage all of you to do service with the Society, and I echo what Dr. Wysham said. It's most important that we take care of the next generation. Thank you. Now, I have the true pleasure to introduce our first speaker, Dr. Genevieve Neal-Perry. Genevieve Neal-Perry is a Robert A. Ross Distinguished Professor and Chair of Obstetrics and Gynecology at the University of North Carolina School of Medicine. She has expertise in the treatment of infertility, fertility preservation, recurrent pregnancy loss, and the development of procreatic management plans for same-sex couples and single parents. She is also a leader in the area of medical management of menopause, PCOS, premature ovarian failure, and other endocrine disorders that affect the function of the hypothalamic-pituitary-ovarian axis. Dr. Neal-Perry conducts human and rodent research that is focused on understanding the nutritional and environmental factors that affect the age of puberty, ovarian and testicular function, fertility, and the age of menopause. She is especially interested in the effect of maternal or paternal environment on fertility of male and female offspring across multiple generations. Dr. Neal-Perry has been a member of the Endocrine Society since 2001 and has served on the Special Programs Committee, the Research Affairs Core Committee, the Board of Directors, as well as the Endo Editorial Board. She has also served as Vice President for Basic Scientists on the Society's Leadership Council. Please join me in welcoming Dr. Neal-Perry to discuss current challenges in fertility and reproduction. Good afternoon, and thank you all for being here and hanging out to the end of the meeting. It's so nice to actually see people, touch people, I've had some, definitely been feeling a little lonely over the last two years. So I'm happy to have the opportunity to talk to you about addressing challenges in fertility reproduction. And what I thought I would do is walk you through a little bit of the history, because I think understanding the history can help you think about where we need to go. And so I'll walk through our history, which has been quite amazing over the last 40 to 50 years. So there's something going on here with the slides, I'm sorry. No, okay, now it's right. Yeah, this is not right. Okay, stop. Yep. We're going to... We looked at it. So let me tell you a little bit about what I was going to talk about. Thank you. You'll see it pop up. Okay, great. So really it was going... So there's been a lot of basic science that allowed us to actually come to the day where we had our first IVF patient. And the first IVF patient was done in Great Britain with Steptoe and Edwards. And it was with a couple who had tubal factor infertility. And with this couple, they had gone to see physicians and really had been unsuccessful. And there had been some work done previously by Pincus and some other folks that had demonstrated that you could collect an egg and you can put the sperm together and you can fertilize the egg outside the actual tube. And so they did this procedure with this couple and they created this embryo. Bring it up. Okay. So this is the deck that we have. Mm-hmm. Yeah, I looked at it together. Can we see it? Okay. So they collected it. So this is when you learn about resilience, right, and activation of the stress axis. So I'm sure my cortisol levels are quite high. All right. Let's see. Yeah, there's something going on here. Okay. That's okay. So they were able to collect this egg and they were able to use the sperm, create an embryo, and then we had the first birth of Louise. And that was an amazing thing. And this is after many years of scientists understanding that infertility was a problem and how do we circumvent this in order to help achieve families for individuals who are not so fortunate. And that really was an explosion point for us in reproductive endocrinology and it was really a springboard. And the wheels just really started moving quite quickly from that point forward. Okay, I'm not sure what's going on with the sides. Do this button. Okay. You want to start it over? Go, okay. Okay. All right. All right. Here we go. Okay. So the history a little bit and then I'll talk about some eminent challenges, disparate outcomes, and future emerging ideas. Nothing to disclose in terms of the history. This is what we started talking about. So believe it or not, the first IUI or intrauterine insemination occurred in 1980, excuse me, 1884. And it was done with a physician named Pan Coast. And he actually gave a young woman anesthesia, put sperm in a uterus that was not her husband's and didn't tell him for many years. So thank God that we have ethics. But that was really the first kind of IUI. Then in terms of sperm cryopreservation, first time 1953, although sperm was cryopreserved, it really wasn't used in the way that we think about it today. Then we had our first IVF baby, Louise Brown, in 1978. This was also followed by the first cycle using clomiphene citrate and ovulation induction. And these are really important advances in terms of where we are today and how we get there. In 1980, there was also some initial work being done by Handyside in terms of cell biopsy and really kind of getting us ready for the concept of pre-genetic testing or pre-implantation diagnosis. All right. Then in 1981, we had our first IVF patient here in the U.S. And Samantha Steele was actually the fourth one, Elizabeth Carr. And that was with the Jones Institute. That was at Howard and Georgiana. They also developed the first IVF lab. And it was, again, really the beginning of a roller coaster ride to where we are today. As the years progressed, we also had our first donor egg cycle and embryo. We started to really think about the process of how do you collect eggs. The initial process was not very efficient. It was done with laparoscopy where the person had a dominant follicle. We went in and collected one egg, and that was it. The efficiency of IVF like that was about 5%. And so the goal was how do we create more oocytes and how do we collect those oocytes in a less invasive way so that we have more of a platform, more embryos, and a great opportunity to achieve pregnancies for couples. As time progressed, we realized that we're also able to generate these supernumerary oocytes and embryos. And so could we freeze the embryo and use those embryos to create life? And we were able to do that. We used what's called a slow freeze technique. It was not the most effective way to do it in that embryos didn't necessarily always survive. And so pregnancy success in our best prognosis groups was about 35%. But that was, again, a landing point that gave us a new opportunity in terms of how we would advance the reproduction and infertility of our patients. Now, in 1985, remember I told you that the way that the egg was collected was using laparoscopy and laparoscopic oocyte collection. We developed a way to actually use an ultrasound in the vagina and use needle guided collection of eggs. And with the advent or use of controlled ovarian stimulation, we were able to collect multiple eggs and have multiple eggs for fertilization. All right. So we've really gotten past the challenge of collecting eggs. But not all eggs would fertilize. And not all men had enough sperm to use the typical IVF process. And so we needed to develop a system where we can either inject a sperm or create an opportunity where sperm can get into the eggs easier. And there was a procedure called subzonal sperm injection. And it was the first step or kind of the pathway for using intracytopathic sperm injection, which allowed us to go from 35% to 40% fertilization to 80% to 90%. The next step in terms of progress that we've made was being able to do genetic testing on embryos. There are couples, as you know, who have genetic disorders that are inherited genetically through recessive as well as autosomal dominant disorders, that if you could identify a way to select those embryos out, that would be a helpful way of really improving fertility. And in 1990, PGD with sex selection was developed by Handicide and also assisted hatching, which was important for being able to collect the cells to actually do the genetic screening of the embryos. In 1992, IVF was advanced further by ICSI, that's intracytopathic sperm injection, remembering Susie was kind of that springboard to the development of ICSI. And then we had our first IVF pregnancy. We also developed recombinant gonadotropins rather than menotropins, which were basically highly purified urinary gonadotropins. And this gave us the opportunity to treat patients without them developing allergies to the medication as well as not getting infections when dried or desiccated pituitary was used instead. In 1993, we advanced the male fertility component through the use of ICSI and a testicular biopsy. And this actually created the opportunity for individuals who had obstructive basal spermia to actually procreate. And with the advent of the genetic testing, that also allowed us to identify individuals who may have been at risk for cystic fibrosis so that not only could we now fertilize these eggs with testicular sperm, but we can also identify embryos that might have been affected. 1995, aneuploidy testing was also introduced by Jacques Cohen. And we also started to use spermatids for ICSI purposes. In 2000, we had our first successful ovarian transplant with Octay and Kalia. 2002, the first pregnancy after a biopsy for pre-genetic testing. 2004, our first pregnancy after an orthotopic ovarian transplant. This was a huge advance. And also, we began to do single-blast embryo transfers. So this was a really important advance because this meant we were no longer transferring multiple embryos and having multiple pregnancies. That was a risk of IVF. And with the advent of single embryo transfer, we actually improved outcomes for our patients significantly. In 2006, our first IVF patient or infant actually had her first child, a spontaneous delivery. And that was amazing because we didn't know. What did IVF actually do to the reproductive outcomes of individuals who were born that way? And that was a really important next step. In 2007, we developed blastocysts from using in vitro maturation of oocytes. And this really is an important step in terms of being able to provide the opportunity for patients who may have a diagnosis of cancer, who may have a diagnosis of cancer and who cannot undergo IVF, but to be able to collect those eggs and actually mature them in vitro was really important next step. In 2010, a Nobel Prize to Robert Edwards for his work. And then we actually started to use human cloning stem cells in 2013, as well as starting using time-lapse imaging for evaluating embryos as a way to select the best. So kind of gave you the history, which has been quite impressive in terms of the speed in which things have happened. But there are challenges, and many of the challenges are related to some of the advances that we've made. So the primary goal of a reproductive endocrinologist is to generate ample embryos and to identify unaffected embryos so that our patients have a normal live birth. So we were able to create these embryos, and we can identify these embryos now. So how do you improve embryo selection? And that is an important part of kind of where do we go next. For those of you who have not seen what embryos look like up close and personal, this is what's called a cleavage stage embryo, it's a day three embryo. This is the embryo that you typically find in the fallopian tube, and this is called a blastocyst embryo. And this is the embryo that's in the uterus at the time of implantation. One of the most significant advances that we made was culture conditions, because we initially used the typical culture that you would use in somatic cells, thinking that was enough for embryos. But being able to understand that the metabolic requirements for cleavage stage versus blastocyst stage embryos helped us identify better embryos so that we now went from using a cleavage stage embryo to a blastocyst embryo, which has a better pregnancy rate. So pregnancy outcomes are significantly impacted by age. And we know that as women get older, the quality of the eggs change and the risk for aneuploidy increases. And we also know that, in terms of live births, that if you're less than 35, you have more live births per number of oocytes as opposed to if you're older than 35. So how were we actually evaluating embryos? Well, it was really by eyesight. It was a grading process. Essentially, with a cleavage stage embryo, what we would do is look at the embryo, count the number of what's called blastomeres. And there are these cells here. We look at these blebbing, or what we call fragmentation. That was not a good thing. We look at multi-nucleation in these cells. Again, not a good thing. And we would grade these embryos and determine whether or not they were good or bad. And this is how we would select embryos. Now, with the blastocyst embryos, we use a different process in terms of evaluation. Again, the embryos are scored based on the way that they looked in terms of the expansion of the embryo's blastocele, as well as the inner cell mass in terms of the way the quality looked and the outer cell mass. Inner cell mass is what becomes the embryo. The outer cell mass is what becomes the placenta. And the embryos are graded one through six based on whether they're expanding or hatching. The higher numbers are better. The more cohesive and the shape of the cells are graded as A, B, and C. And so this is the way. Again, all visual. This was really helpful, but it wasn't perfect. So in terms of trying to understand a little bit more about how we might be able to select for the best embryos, we started using genetic evaluation methods. We use FISH. We use comparative genomic hybridization, as well as array CGH. And what we found was that we were able to select for some embryos that had a better quality, or I should say that were a euploid, but it didn't actually equate to necessarily more pregnancies. So the limitations with the biopsies at day three was that we would just biopsy one cell, and that one cell would be used to determine the entire embryo itself. There was lots of mosaicism. We saw reduced pregnancy rates. And it really wasn't shown to be very beneficial. So we actually stopped doing it at that stage. Then we actually started to do what's called trophectoderm biopsy. And we saw better survival rates. We were able to biopsy more cells, five to six cells rather than one. The challenge, though, was that it was a timing issue. You had to do day five, day six. We still saw issues with mosaicism. And fewer embryos actually make it to this stage. And that, lastly, that the trophectoderm may not necessarily reflect the inner cell mass. So the point is that not all that glitters is gold. There was lots of inter-observer variation in technique. Cleavage versus blastocysts. Certainly, what was graded as a good cleavage stage embryo was not necessarily one that made it to a blastocyst stage. And we really could not and still are not really able to select out for the best embryos. So where do we go from here? There have been several studies looking at the spent medium in which the embryos grow, looking at proteomics, metabolomics, as well as a free DNA release from the embryos as a way to evaluate in a way that does not actually involve biopsy in the embryo, so in a non-invasive way. And while these studies are promising, we're still not there yet. So this is a gap in terms of identifying the best embryo and being able to help individuals achieve pregnancy. So as I said earlier, it's all about the egg. We know that age and egg are the best predictors of pregnancy outcome. So how do we get from having one golden egg to having multiples? And that is one of our challenges in infertility treatment today. The older you are, the more eggs you require for a live birth. And this is just a figure here showing the number of eggs needed for a live birth relative to age, 18 to 34, up to 40. And you can see that the younger you are and the more eggs you have, the more likely you are to have a pregnancy. Whereas the older you are, at some point it doesn't really improve your outcome by having lots of eggs. And that has a lot to do with aneuploidy development. So are there opportunities to actually evaluate the egg itself before an embryo is created? There have been some studies where they've done polar biopsies. And a polar body is the extruded portion of the egg after it's gone through meiosis. And although there have been some data to suggest that you may have some parallelisms in terms of what the egg's ability to result in a live birth might be, it has not been, again, a golden process. And it's not something that we're able to use at this time. So having a way to evaluate the egg before it's even an embryo is really, again, a gap in reproductive endocrinology. So one of the things that we know happens with aging is that the mitochondria are not as robust, and they're not as functional. And so there have been some studies looking at the transfer of mitochondria from other oocytes that we know that are healthy from young women or from other cells within a woman's body that have normal mitochondria function. And there have been some studies that have shown that that has actually improved outcomes. There are also studies where you remove the entire spindle apparatus as well as the nucleus of the egg and put it into an empty egg. And that has been shown to have some promise. But again, we're not there yet. And the transfer of the nucleus as well as the spindle is not something that's legal here in the United States. And there are very few places that actually you do mitochondria transfers. So what about the opportunity for in vitro gametogenesis or gametogenesis with stem cells? That seems to be something that's quite promising. Again, we're not quite there yet. But there have been some studies showing that human-induced pluripotent stem cells can generate oocytes, and they can generate sperm. The challenges with it is that what we've learned is that there are other things about the ovary that are important for egg development as well as within a testes so that when we use these gametes, we don't yet have the same level of fertilization outcomes that we have in using cells that have not been created this way. But this is something that's on the horizon and an opportunity for the future. So one of the biggest challenges in infertility is really access and disparate referral patterns. What I want to show you here, and I know this is a little bit hard to see, but this is a figure that basically shows what's called a density plot. And what they're doing is they're asking patients, how easy is it for you to do different things to see your doctor? And it's based on your income. It's based on race. And what they have clearly shown is that there are differences in terms of the referrals of African-Americans and Latinas to infertility specialists. In areas where you do not have mandated care, we also do see disparities in terms of people who are either lower socioeconomic status as well as individuals of underrepresented in medicine. And health care disparities also have less access. So obesity and reproductive outcomes is one that has also been shown to be a challenge and possibly something where there's intervention as a possibility. What this figure here shows you is a cumulative likelihood to live birth based on age and based on your BMI. And you can see that no matter how old you are, if you're heavier, your chance for a successful outcome is significantly different. And that's separate from any of the consequences that are associated with being obese during pregnancy. But we do know that obesity by itself is a modifier of fertility outcome. There have been studies that demonstrate or suggest that this may be related to inflammation, the inflammatory environment of obesity. So I kind of suggested that there are racial disparities in infertility and IVF outcomes. And I'm just going to go through a few slides here. One of the challenges that's been reported is that African-American women in general are not receiving assisted reproductive technologies until later in life when they're already disadvantaged because of their age. Again, some of this is related to resources, but also there are studies that demonstrate it's related to referral patterns. In terms of BMI, one of the known modifiers of success is a BMI greater than 30. African-American women tend to have a higher BMI. And that may also contribute as well as they're more likely to have an abortion, a miscarriage, and also more likely to be nulliparous. In terms of IVF outcomes, important to notice is that AMH or ovarian reserve is not significantly different. The mean oocyte count is not different. The number of embryos that are acquired preserved, the risk for diminished ovarian reserve is not different. However, there are significant differences in terms of implantation rates, the risk of not being pregnant, the risk for an abortion, as well as their reduced likelihood for live birth rates per cycle. In terms of fertility diagnosis, the fertility diagnosis most commonly seen in African-American women are tubal factor or uterine factor. This ovarian reserve modifier is really in terms of the response. What you see is that African-American women may require more medication, but that also could be related to obesity. There is also, when you look at whether mandated in terms of fertility access, if you look at utilization in mandated versus non-mandated states, you can see in states where there's mandated fertility services, you see more African-American individuals were using services, as opposed to in states where there's not. There is this disparity. So I'm just going to show you a little bit of research that we did along the lines of trying to understand the racial disparities in IVF. And one of the things that we looked at was actually on vitamin D. And so black race is associated with an unsuccessful outcome with assistive technology. But again, it's poorly understood why. It's not because there are fewer eggs. It's not because fewer embryos are generated. But what we do know is that there is an increase in age, as well as obesity. And these are outcomes that are associated with poor outcomes. And so what we did in this study here was to see whether in individuals who have poor outcomes, was there also an association of vitamin D deficiency? And so it was a retrospective design. And we compared African-American women and Caucasian women. And it was a group of individuals who had undergone IVF. And we looked at pregnancy outcomes, as well as serum outcomes. And what you can see was there was no significant difference in age. There was a significant difference in BMI for African-American women being slightly heavier, but with a wider range. The number of eggs not different, as I had suggested, has already reported. Vitamin D levels were significantly lower. And what we found in individuals who had vitamin D levels that were lower, they also tend to have fewer pregnancies. And what you see here is in BMI, vitamin D. And was there a clinical pregnancy? Yes, for African-American if the BMI was around here. If they were vitamin D deficient, they did have a clinical pregnancy. But if they were vitamin D deficient, there was no clinical pregnancy. And that was also true in looking at the biochemical and, sorry, clinical pregnancy. So the summary here was that overweight African-American women with vitamin D deficiency who undergo IVF are also at increased risk for having a poor outcome. We then took this to the lab to try to understand a little bit more about vitamin D deficiency and how it might affect fertility. And we did these studies in mice where we induce vitamin D deficiency with a diet. And these were C57 black female mice. And what we see in our controls that we saw a smaller litter size in the mice that were fed a vitamin D deficient diet. We then went on to ask questions about whether it was poor egg quality, poor embryo quality, implantation failure, trying to understand a little bit more about what might be going on in African-American women. And what we did first was to look at placental site nodules as a way to determine whether there were implantation defects. And what you see here are controls. And then here, this is what we were seeing in our individual mice that were exposed to vitamin D deficiency. So we saw fewer implantation nodules and suggesting that there is a defect either for implantation or that they had fewer eggs. We looked at the corporal lutea, which would be a surrogate for the number of eggs in the ovary. And we saw that in the vitamin D deficient animals, they had just as many, if not more, corporal lutea than the controlled animals. So why is this happening? Is it a uterine factor? Is it an ovarian or oocyte problem? And then we asked the question about whether they're just making fewer eggs. So we did a controlled ovarian stimulation in these mice. And what we found was that in the controlled ovarian stimulation in the vitamin D deficient animals, they actually created more oocytes. So it wasn't an oocyte issue. We then went on to look at whether it was for oocyte quality. And in this study, we did a TriPAN blue exclusion study looking at the number of alive oocytes that were collected in control versus vitamin D deficient. And we saw that the vitamin D deficient animals had fewer live oocytes. And then when we looked at the actual eggs themselves, what we noticed was that there were spindle defects and that the eggs generally looked as if they were breaking down. So we think that the vitamin D deficiency itself may be contributing to poor oocyte quality. And that may be one of the ways that we see some differences in poor outcomes in African-American women. So in conclusion, fertility treatments have made it possible for many to procreate and preserve their fertility. There are disparities in access and outcomes that limit just how successful we can be and claim to be as fertility specialists. Emerging technology should be directed towards making treatments that are more affordable and improving pregnancy rates as well as pregnancy outcomes for all groups. So with that, I'd say thank you. Thank you, Dr. Neal-Perry. Next, we're going to hear a prerecorded talk from Professor Jacob Hanna. He earned his BSC in medical science and PhD in immunology, MD in clinical medicine summa cum laude from the Hebrew University in Jerusalem. He conducted postdoctoral research at the Whitehead Institute for Biomedical Research at MIT, and is a professor of medicine at the University of New York Whitehead Institute for Biomedical Research at MIT, and joined the Department of Molecular Genetics at the Weizmann Institute in 2011. Professor Hanna is pioneering techniques in induced pluripotency, artificial embryo models, and reprogramming of adult cells. Induced pluripotent stem cells have regenerative properties that are almost identical to those of embryonic stem cells, but can be created from adult cells without using an egg or fetal material. He was a lead researcher in a study that showed how further modified iPS cells could be used to treat sickle cell anemia in mice, the first proof of concept of the therapeutic application of iPS cells. He has uncovered novel pathways regulating the reprogramming process, and the first to derive pristine, naive human pluripotential cells equivalent to those derived from mice, and can generate cross-species humanized chimeric mouse models that have human-derived tissues. He was the first to expand prolonged periods of advanced and normal mammalian embryo development in an artificial uterus environment outside of the maternal womb. And in addition to demonstrating the power of cell reprogramming, his work offers a promise of powerful new research models for cancer, degenerative disease, and infertility, as he pioneered generation of human progenitor cells of sperm and eggs from iPS cells. I'm pleased to introduce Professor Hanna's talk on ex-utero systems for mammalian reproduction. Please turn your attention to the screen for Professor Hanna's presentation. Good afternoon, everyone. First, I would like to thank the organizers for inviting me to present in this great conference. I apologize that for personal health reasons, I could not make it in person. And I wish you all a very enjoyable meeting. What I'll talk about today is about ex-utero mammalian embryogenesis and trying for understanding stem cell biology and early development. First, I shall my conflict of interest slide declaration. My lab focuses on understanding pluripotent stem cells and very early development. We are very much interested into all processes that accompany early development from fertilization of the zygote until the blastocyst formation, implantation happens, the epiblast forms into an egg cylinder shaped one in the mouse, and then even some cells are specified into primordial germ cells. And the remaining of the embryo goes on to make the embryo proper. And these processes are accompanied by very rapid and dramatic changes that we are limited in understanding because even in the mouse, we can get very little material in these early stages. As well as even with single cell technologies, we are very much limited still to perhaps RNA sequencing. We cannot do biochemistry. We cannot do easily perturbations. Of course, when you start to talk about other species than the mouse, like not to mention humans, this becomes very complicated and it's very hard for us to study. And another side of the coin of studying this early development is also the fact that we can derive pluripotent stem cells from early embryos. And these cells can be derived from early embryos from pre-implantation stages and from post-implantation stages. My lab has worked on characterizing these cell types both in mouse and humans. And we now know that the pluripotency has different flavors. So it's not a binary zero versus one state, but you can have these naive, very early pluripotent stage, which more corresponds to the blastocyst pre-implantation. For example, in females, exon activation has not started. Global DNA methylation levels are very low in these cells versus once later stages, what we call prime pluripotent stem cells. So they're still pluripotent. They have not differentiated yet, but they're about to. And they're in more advanced stages. For example, exochromosome activation in females already happened, DNA methylation increases, and so forth. So there's a lot of differences, but really the field and the question we ask, you know, who cares? Overall, they're both stem cells. They can differentiate all cell types. Should we, does it matter at all? And I would say one of the first proofs that it does matter came from the work of Mitinori Saito about 10 years ago trying to make PGCs, primordial germ cells, from mouse stem cells. And he was really the first to make authentic PGCs, and if you dig deep into why did he succeed, he had to start with naive pluripotent cells, and these are grown into these with the inhibition of ERG signaling. He had to give them priming for two days, and then he can give them BMP4, which is the major cytokine, and he can get these PGCs, this double positive population. So he had, it works very well, but he had to make sure he carries out the sequence of event like in development from naive to primed to PGC induction, and it really matters. Actually, our group afterwards tried to, and others tried to reproduce these findings in humans, and can we make human PGCs? And actually this protocol by Saito failed, but the reason for that was that human cells that were available at the time were not in naive conditions. So my lab has been developing how and which factors we need to induce to keep human pluripotent cells in this very early stage, and I will not get into the details and just say that for us the major divider between naive and primed is the ability of the cells to tolerate inhibition of ERG signaling, meaning naive cells actually like and expand better once ERG signaling is inhibited. And my group really has identified recently the different combination of cytokines to make human naive pluripotent cells, and we've published different versions that we continue to improve them over the time, but really we've now reached the stage that also in humans we have naive pluripotent cells and human pluripotent cells. And once we had actually these naive pluripotent cells, we could immediately make human PGCs like was done in the mouse, meaning that the failure before, it wasn't because it was a different protocol was needed, but actually the starting material. So if you start with the human naive cells, you can give them BMP4, and you can see with these reporter cells in the FACS plot, we can get very efficiently and very quickly human primordial germ cells. So yes, there is a difference between naive and primed cells, and it's a functional difference that we do not understand yet. And questions that remain unanswered, which is, for example, why do we have to start with naive cells? Why is priming essential for making PGCs? And also if we induce too long priming, meaning a week instead of two days, the competence for making PGCs goes away. So there is really something happening about a competence window here that's being opened up that we do not understand and we feel it's very important to do because it also could be relevant for other lineages. This has brought us really to the current theme of work that we're working about, embryos and artificial embryos, but my point is that we'll start, this starts with asking that we're talking about naive pluripotency and primed pluripotency, and I want to understand what is the functional outcome of these things. We have to, the functional readout is basically the embryo. So we do not have a system either in vitro or in vivo where we can continuously, for example, take a blastocyst, make it advanced to become into a post-implantation embryo, let's say we make a mutation and follow the outcome. So we don't have this continuum happening in the petri dish. Therefore, we are very limited into asking about the questions, what is the importance and consequence of moving between naive pluripotency to primed pluripotency and then organ formation. And this block really obvious, which we call is the uterine barrier, which is faced in basically all mammals, where we can very efficiently grow mouse and human embryos until the blastocyst stage. And this is of course the basis for IVF treatments. And once we reach the blastocyst stage, we have to implant back into a surrogate uterus, this blastocyst. We cannot grow and move beyond the implantation stage. And this is very important for us because the drama of organ formation happens right after implantation. So for example, in the mouse, this happens in the first four days after implantation, we have symmetry breaking event, we have early gastrulation, late gastrulation, and then we start the organogenesis processes that ends at day 13 and a half. So we have basically 75% of mouse pregnancy happening inside the uterus, which is inaccessible, it's non-transparent, it's in vivo, we do not control the environment, and it's very hard for us to see these very dramatic events happening that even happen when the embryo is extremely small. Therefore, we started thinking about can we make a solution to grow advanced embryo a little bit more, maybe for one or two extra days beyond the blastocyst. And this is what we call exutero-embryo culture. Now, gastrulation is a very important process in development. You might know the famous quote by Lewis Wolpert about what is the most important day in our life, where he said it's not birth, marriage, or death, but gastrulation that is truly the most important time in our life, because if we don't gastrulate correctly, we end up with a lot of problems and a lot of birth defects. And there's also another question that we are trying to ask here, which in principle, can mammalian gastrulation, at least in one species, be fully captured exutero? And can mammalian organogenesis be fully captured exutero? And can we actually capture both in companion? Can we continuously capture gastrulation organogenesis outside the utero and really show that the uterus is not needed for the inductive signaling and the patterning of the mammalian embryo? So, why we also would like to do that, as I mentioned, we cannot grow embryos outside the uterus at advanced stages. For example, even if we take a post-implantation embryo from the uterus, we cannot put it back into another uterus. So, this really prevents our ability to do this experiment that I described. We want to take an embryo, mutate it, and follow over the time the consequence of this mutation. And of course, also the environment and liquids and the metabolites in which the embryo is growing inside the uterus is not something that we can easily control. And that's why we set out on this attempt. And really, if you go back about 100 years ago, there were very, very few attempts to grow mouse embryos outside the uterus in advanced stages. Mostly, these experiments were able to reach embryos growing for only about 24 hours of development, let's say from day 8 to day 9 or day 9 to day 10. They could not capture gastrulation. They were extremely inefficient. Most embryos were abnormal. Therefore, these settings were not widely adopted. They were very good, for example, for very crude kind of experiments. If you want to induce an ectopic head or an ectopic limb, so you don't really need a normal embryo. But what we are trying to aim here is to capture efficient and normal development in mammalian without delay. So, we started thinking to build a device. And we thought about it in a way like a ventilation machine. We don't want to ventilate the embryo lungs, but rather the environment surrounding the embryo. And we started thinking that we need to have an incubator part. We need to have a controller device. We need to have a gas mixing box where things get mixed and measured before they are flown to the incubator. And then, over a process of years, we started designing each of these components, which are the parameters, which are the sensors, what are the things we need to be monitoring. And slowly, as we try to improve this process, we've learned what are the parameters that are needed to control. And so, basically, what happened, we were able to be able to grow for one more day, and then two more days, and then five more days, and of course, with increasing quality and efficiency. And this is the final product that I show you here. This is the controller device that we have. It's not a pretty one because we make it in-house, like it's like a garage-made unit. But it is then connected to the gas mixing box that I mentioned to you. And the other part, which is the incubator that is connected and controlled by this device. We've learned that we need to carefully regulate oxygen levels. For example, we cannot give permanently 20% oxygen. We've learned that hyperbaric pressure is critical. And this kind of makes sense because inside the uterus and the embryo environment is slightly grown into higher pressure. That we need to grow the embryos in a dynamic glass bottles that are spinning because the embryos are very, very adherent. And once they adhere, they become distorted. Phototoxicity, we have to put a black cloth or a diaper. Otherwise, the embryos will not be abnormal. I'm talking about for prolonged periods of time. It's not that if you're working with for half an hour, you don't need darkness. But you need to cover the incubator. And of course, inserting a humidification unit and so forth. And this is when the device is working. Each bottle has an embryo that is swimming in the fluid, in the liquid that we had to develop to nurture these embryos. And basically, this media is composed from a combination of both rat and human serum and extra need for high glucose. And I want to also emphasize that it's very critical to have freshly isolated serum that doesn't have byproducts of hemolysis. If there's some hemolysis byproducts, it's very toxic for the embryos. And you can see here in this video, when we start with seven and a half embryo, which is late gastrulation, within one day, we get yolk sac expansion very dramatically. You can see the head and the legs. And after three days, you can see the embryo turning with the heartbeat. And you can see the blood, which is this is the fetal blood, the fetal hematocyte system. And they reach, at day 11, one centimeter in size. And then the embryos die, basically, from hydrops fetalis, because they are dependent on diffusion for their nutrition at the moment. So just to show you, we had to develop an entire toolkit of how do you set up the machine? How do we measure the pressures? How do we calibrate it? How do we feed the embryos? For example, we move them from one bottle to the other into a new liquid, and then mount them again on the machine. And these videos are on our website, as well as on Joe, to kind of describe the techniques surrounding this work. And just to show you that, if you look here now that once we have the calibrated conditions, which we refer as controlled, you can see that if we don't give high pressure, you see how we get abnormal embryos. If we don't give enough glucose, we have abnormal embryos. If we do not have a gradual increase in oxygen, we will have abnormal embryos. So the process is very, very efficient, but it needs to be done with accurate parameters. Then we started really just characterizing side by side in utero versus exutero embryos, dissected and undissected. And just to show you that these embryos do not have delays, they have normal size and morphology at very, very high efficiency, for multiple strains. We've also done atlas of 12 different markers. And as you can see here with the slide sheet microscopy, there is no spatiotemporal abnormalities, there is no delay in gene expression in these embryos. But actually, we didn't stop there, because as I told you, we're starting with day seven and a half, which is late gastrulation. And we want to start from pre-gastrulation stages, which is E5.5 and E6.5. So we developed another protocol, which is a static protocol. We use the same media we developed before, but now we use these IBD plates that are typically used in IVF units. And you can see very efficiently gastrulation can happen in these static conditions. You can see now in these videos, this is embryos that are labeled with red. And you can see how in 58 hours that yolk sac grows dramatically in these embryos. You can see the primitive streak moving and somite formation and then head fold formation. And this just shows the power that we can now visualize normal development under the microscope and with very high efficiency. As I mentioned before, then we could combine them, we can grow from day five to day 11. And these embryos basically really capture gastrulation and organogenesis outside the uterus by combining the static and the dynamic conditions. And just to show you to look at an unbiased way that these embryos are normal, what we do is single cell RNA-seq analysis where we take embryos and look at all cells to make sure that we're not overlooking a marker or a population. And you can see here that if you overlay the X utero versus N utero on top of each other, they're nearly identical. There are very minimal changes between these embryos and that we can capture all lineages and cell types that are found in these embryos at this stage. There is, as I mentioned, very minimal changes. You can see here at the late stages, there's this cardiomyocyte gene expression change, very mild, and erythroid change. And this is, as I mentioned, because of placental insufficiency, hydrospitalis, because the embryo is starting to compensate with a cardiac heartbeat and increased red blood cell formation. So this is really consistent with this insufficient oxygenation at day 11 in these embryos because they're now too large to rely only on diffusion in this system. So to summarize, I've showed you that we can grow the embryos and you can see here not only the change in the shape of the embryo but also the change in the size. And I want to emphasize that I like to give the credit to the embryo and I think these findings underscore the strong self-organization potential of the embryo and that the embryo has its own program and that if we just give it the right conditions and the nutrition, this program can proceed outside the uterus correctly. And that's why I do not use the word artificial uterus for many reasons but one of them because I would like to give the credit more to the embryo itself. And I feel that we are just unraveling the potential that it already has under the right conditions. Now as we're trying to develop a research tool, we don't want to just grow embryos but we want to be able to manipulate them and do a lot of functional assets. So one of the things we would like to do is rapid mutagenesis, basically take an early embryo, put a lentivirus that causes with CRISPR-Cas9 a mutation, and then grow the embryo outside the uterus and follow the outcome. So for example we develop a protocol where we can inject into the pro-amniotic cavity one microliter of a lentivirus, in this case I show you GFP, NSN-GFP, and grow the embryos for five days and you can see how quickly the entire embryo is labeled with this method and the embryos grow still normally with the same efficiency, showing that really we can now do rapid mutagenesis of embryos ex-utero. Since we have now the embryos growing outside the uterus, at any time point you can take the embryo and use this media and mount the embryo on the microscope and visualize a certain organ formation. So here you're looking at an embryo just as the neural tube is closing and we take it at day nine and then we start imaging and you can see this characteristic kind of zipper kind of movement of the neural tube closure, again showing that this is now enabling us to look even at more higher resolution organ formation in ex-utero. Another example just to show advantages, since these embryos are grown inside a bottle we can start doing teratogen testing and to show that the environment can induce different behavior or defects in these embryos. So if we add valproic acid we immediately in the next day start to see open neural tube defects that are expected with this drug, again showing that we can recapitulate known phenotypes now in a more controlled environment. So this has been exciting for us and I hope that I convinced you that we make now one step further into more dynamic systems that enable us to look at tissue mechanics in the embryos, teratogenic studies, lineage tracing under the microscopy, and really when we combine with very advanced live imaging as I show you here we can look at the entire embryo doing very critical processes outside the uterus in critical stages of gastrulation and organogenesis. We also can use these embryos for chimera studies, meaning we can inject a human or a mouse cell population and follow how these cells migrate, integrate, and if they fail why does that happen. And with the rapid mutagenesis protocol that I mentioned to you is we can now start dissecting you know genes that are critical for mouse gastrulation or specific organ formation in a much more accessible way than we could do before and not rely on just genetic snapshot studies that of course we've learned so much from them but they are limiting and they are time consuming. But then this now opens the door on a very new field of research which is called synthetic embryology and I feel that this field was completely blocked because it lacked techniques like the ones we developed before and I'll explain. Everything that I've showed you so far is basically natural embryos. We can take them out at day five or day six or day seven and grow them outside the uterus. However as you know we can grow embryonic stem cells or iPS cells in the dish. We can even inject them into other mice and we can see them contribute to tissues. But the question is can we make an entire embryo only from pluripotent cells without using a host blastocyst or a host zygote? And this type of research was blocked because if we lacked the tools to grow natural embryos outside the uterus how are we going to grow synthetic embryos? And we're happy now that basically these results that I showed you now become the reference positive control so we now know what it takes and what is needed to grow mouse embryos during gastrulation and organogenesis and ask if we take now stem cells and aggregate them in a certain way with different mixture of cells, what will happen if we put them in our devices? Can we get embryos only from stem cells? And this is what's called synthetic embryos. Can they be will they be natural embryos and or normal embryos and can they also constitute a novel model for looking at cells and cell formation inside the embryo? And this remains an open question but we're very excited because this is really these platforms that I described to you opens a new avenue to really start to improve and test such synthetic embryos from also different species. With that I would like to finish and thank my team members. This work was led by two great PhD students from Mexico, originally Alejandro Aguilera, Bernardo Oldak, and two staff scientists from Israel, Radha Massaru and Noah Norvershtern. We have a great collaboration with Rambam Hospital, Dr. Itai Maza and Dr. Nader Ghanem on help on getting umbilical cord serum that is often needed for for growing these embryos ex-utero. And and the different members and collaborators that really helped us to to conduct the studies and the funding agency. And I'd like to also finish and just pitch that you know my lab is always looking for very outstanding PhD students and postdocs and if any of the topics are of interest to you I highly encourage you to reach out to me and inquire about position and discuss science and cool things we we can do. Again I thank the organizers and thank you for inviting me. I want to thank you for today's speakers, award recipients, and for our audience and I hope you enjoy the rest of your time at Endo. Thank you for coming. I do want to announce that the next two sessions back to back in this room will be the year in nuclear receptors, the top 10 reasons why it's cool to study nuclear receptors, followed by the clinical year and review all things adrenal by Dr. Hammer. So I hope you'll stay and and enjoy these two very uh in very interesting talks I'm sure that are that will follow. And again wish you all the best, wish the best to Dr. Kaiser, and look forward to seeing you folks next year in Chicago.

ENDO 2022

fertility challenges

reproduction challenges

Dr. Rita Cugliani

Outstanding Public Service Award

Dr. Simon Rhodes

Sidney H. Engbar Distinguished Service Award

stem cells

fertility preservation

reproductive endocrinology