So welcome to our session on developmental origins of the endometrium omics from within the cradle. And I am Andy Babu from Rutgers University. And I'm Julie Kim from Northwestern. Great. And we have a fantastic lineup of speakers this morning. And as I was telling them this morning, for me, this is like Christmas, because these are people who have followed all my career. And it's just wonderful to have them. Our first speaker, unfortunately, his flight was canceled yesterday, Dr. Thomas Spencer. But he was gracious enough to record his presentation. He's going to be the first presentation. And after that, it will be Dr. Lessie, and then Dr. Carlos Simon. So without further ado, let's get started. All right. I want to thank everybody for listening to this presentation, both in person as well as virtual. I'm sorry I couldn't be there due to some travel-related issues. But nonetheless, it's good to share some exciting new data on endometrial epithelial organoids and the biology behind their use, as well as potential applications. So I'd like to thank some members of my current lab, as well as former lab members, Constance Cimiteris and Harriet Fitzgerald, whose work I'll present a lot of today, as well as close colleagues and collaborators at Mizzou, including Andrew Kelleher and Danny Schuse. And I appreciate funding from the National Institutes of Child Health and Development that support this work, as well as the USDA National Institute for Child Health and Development. So the reason that we study uterine glands is because they're pretty exciting. They're a classic case of regenerative stem cell biology, where the functionalis endometrium is shed and then regrows before and during the polypter phase and undergoes a secretory differentiation with the onset of progesterone production by the corpus callitrium. And these endometrial organoids are And these glands have a role in embryo survival, embryo implantation, conceptus growth and placental development. They also are involved in major diseases of the reproductive tract in women, including infertility, endometriosis, adenomyosis and adenocarcinoma. And ultimately, it's thought that increased knowledge of these glands and how they function are important for biomarkers of fertility, as well as developing stem cell therapy approaches to treating certain conditions such as infertility and recurrent early pregnancy loss. So this slide shows early human embryo implantation. And the purpose here is just to demonstrate that there's ample cellular interactions between the glands and the developing decidua as well as the developing placenta. And you can see here at the trophoblast plate stage, you have an inner cell mass with a Zincidio trophoblast that's opposed to decidualizing stromal cells right next to a uterine gland. And then this picture provided by Alan Henders at UC Davis shows direct implantation of the developing placenta and embryo into the mouth of a uterine gland. And so this area of early conceptus maternal interactions is replete with interactions between the glands, stroma, as well as the developing Zincidio trophoblast. And indeed, glands are thought to be very important for the entirety of the first trimester of human pregnancy. So as early as 10 days, you can see here the embryo marked as EC on a hypertrophic bed of uterine glands. And we know that secretory products of the Zincidio trophoblast, including HCG, as well as placental lactogen, or CSH1, cause this hypertrophic response. In this four-week embryo, you can see this section of the developing embryo, the famous Faulkner embryo. That's pseudo-colored blue. And note that this is, the embryo and developing placenta are on a virtual bed of endometrial glands. And their histotrophic secretions, which is pseudo-colored yellow, are actually delivered into the developing intervallous spaces of the placenta. And indeed, the work of Graham Burton and Bertrand Hufford and others have found that the uterine glands empty their contacts directly, contents directly into the developing intervallous spaces. And this is likely very important for nutrition throughout the first trimester, as the spiroarterials are plugged, and you don't have direct content of maternal blood into the intervallous spaces of the developing placenta. We have used, for a number of years, a variety of different animal models to study the role of uterine glands in pregnancy. So here is shown the uterine gland knockout model in sheep. This is created by exposing neonatal ewes to a non-metabolizable form of progesterone that prevents development of the glands in the neonatal uterus. So you can see a normal control animal has literally hundreds of gland cross-sections, whereas the uterine gland knockout has none. And indeed, we found through a series of studies that these are infertile due to recurrent early pregnancy loss. This was the first demonstration that glands were absolutely required for a pregnancy establishment. We subsequently transitioned to a number of mouse genetic models, and the sum of this are the concepts derived from these studies of our lab as well as others are that you have glands that produce lyft under the influence of nitratory estrogen. That lyft acts on the luminal epithelium to allow it to be receptive to triple blast attachment. But we also think that there is a variety of products driven by estrogen and progesterone and under the control of a FOXA2 transcription factor that also likely impact decentralization. And you'll note this in 3D rendering of a mouse implantation site from the work of S.K. Day and Ricola Aurora demonstrate ample uterine glands at the embryo implantation site as well as direct gland duct access to the developing embryo. And so we have in part studied the FOXA2 transcription factor because it's a pioneer transcription factor that can open and gauge chromatin. It has conserved expression in the glands of the uterus across all studied mammals. It's actually a tumor suppressor gene implicated in development of endometrial cancer and endometriosis as well as other human cancers. And importantly, conditional deletion of FOXA2 using the PR-CRE mouse model ablates uterine gland differentiation in the DNA and produces an adult uterine gland that knock out phenotype. So you can see here in the PR-CRE there's absolutely no urine glands. And Jaywook Jiang and Franco DiMaio showed that these animals are infertile due to defects in blastocyst attachment. So we've used this mouse model as well as another one called the Lactoferrin CRE. The Lactoferrin CRE model of S.K. Day turns on after puberty. So it turns on after gland differentiation and will conditionally ablate genes. And so in both of these models, actually they fail to produce lift. They have defects in blastocyst implantation as well as stromal cell decentralization. And so several years ago we simply asked the question, can we give them recombinant lift and cause a pregnancy establishment? Is this the sole factor that the glands produce that are important for pregnancy establishment in mice? And so we used treatments based on original work of Colin Stewart who showed that this administration of recombinant lift on day three and a half and four would rescue the embryo implantation defect in lymph node mice. And what we found is that indeed we could rescue implantation. So on gestational day five and a half there were normal numbers of implantation signs. But by gestational day nine and a half they had been lost in the glandless PR-CRE FOXA2 conditional mice, but were retained in the gland contained lactoferrin-CRE FOXA2 conditional knockouts. And indeed you can see on this gestational day 10 all the embryo sites have been lost in the glandless PR-CRE FOXA2 conditional knockouts. Whereas we had normal pregnancy maintenance, two term with no difference in pup weight or litter size in the FOXA2 conditional knockouts that had uterine glands in the lactoferrin-CRE model. So these were some of the first studies to really demonstrate that uterine gland products are requisite for pregnancy establishment that are other than just lift. Indeed we went on to show in a series of studies that as early as gestational day six there were defects in decidualization genes such as BMP2, BMP8A, as well as WIMP4. So these were absent in the lift replaced glandless PR-CRE FOXA2 conditional knockout mice. And indeed, although we saw normal primary decidual zone formation, the secondary decidual zone was compromised in the glandless mice. We think this is likely due to a differentiation failure. And subsequently we saw apoptosis of decidual cells in the embryo by gestational day eight with complete resorption and pregnancy failure by gestational day 10. And so to translate these mouse findings to the human, we have turned to in vitro models of the human endometrium. And this was reviewed recently by Harriet Fitzgerald in our laboratory in a publication in biology reproduction. And we know that there's a long history of people trying to look at human epithelial and stromal cell function in vitro. But most importantly, about five years ago, Margarita Turco and Graham Burton, as well as Hugo Fang, Colicom's labs, published in 2017, the development of endometrial epithelial organoids, or I may also term this EEO. And this really stemmed out from some work that was predominantly done with intestinal organoids by Hans Clevers' lab. And indeed, in the 2019 paper in nature cell biology, Hugo's lab also showed that you could derive organoids from women with endometriosis as well as endometrial cancer. And this could be used for clinical heterogeneity and drug screening. And so in our own lab, we used the Turco and Baretto methods to develop human endometrial epithelial organoids. The source can be utero biopsies, termed placenta, or menstrual tissue. Can use either normal as well as disease endometrium. Can be obtained from cryopreserved endometrium. They are clonal origin and genetically stable as well as hormone responsive. So in our hands, we dissociate and isolate clumps or individual endometrial epithelial cells and we place them in culture for a period of four days in an expansion medium containing Wnt as well as other stem cell type factors and the epithelia are actually embedded within a major gel extracellular matrix drop. As you can see over an ensuing period of 12 days, they develop these round organoids that are FoxA2 positive. So most of the cells are actually FoxA2 positive as they are in the human endometrium. They express catherins as well as tight junctions and have an apical, an inside apical surface with a basolateral surface on the outside and they also divide as evidenced with this Ki67 stain. As noted in the treatment regimen of two days of estrogen after organoid formation and then six days with either nothing, estrogen, estrogen plus MPA as the progesterone receptor agonist or estrogen, MPA and cyclic AMP. In all of these cases, estrogen up-regulates estrogen receptor alpha or ESR1 and progesterone receptors whereas the combination of estrogen and MPA or inclusion of MPA will down-regulate both the estrogen receptor alpha as well as the progesterone receptor and this is actually what you see normally the change between the proliferative and mid-secretory phase endometrium where the mid-secretory phase is dominated by progesterone and you have a loss of estrogen receptor and progesterone receptors with secretory differentiation. We subsequently did single cell analysis and you'll note that there are proliferative cells, un-ciliated cells, un-ciliated cells as well as a stem cell population and treatment with estrogen actually increases the number of ciliated cells quite substantially as you see both in ambivo as well as in some other published studies on endometrial organoids. If you treat with both estrogen and MPA or in this case we're kind of using a low dose of cyclic AMP to stimulate secretory transformation in each of these cases you also see an increase in ciliated cells as well as secretory cells. And so the functional ability of these to differentiate and response to hormones are intact and is denoted by the complexity of the different cells within the endometrial epithelial organoid and basically mimics what we see in vivo particularly through some new single cell and single nuclear RNA-seq analysis of the human endometrium. So recently we modified our treatment regimen where we only use expansion medium for about six days and then we changed them to a base medium without the Wnt and stem cell factors and so the organoids are still in a major gel drop but they simply don't have the differentiation factors that allow for expansion and then we treat them with either a control vehicle or estrogen or estrogen plus MPA or MPA alone and also started substituting PGE2 for cyclic AMP because Peter Bogner's lab has noted that PGE2 may be a natural mediator of the sigilization that's conserved across mammals. And so we've done RNA-seq analysis. You'll note a robust responses about the up and down regulation of genes by estrogen note an increase in progesterone receptor with estrogen and with MPA you have upregulation of classical progesterone responsive genes including ENPP3. Now what's interesting is that this is of course three donors all together in an edge R analysis but we do see distinct donor or patient specific effects on expression of genes and that either they aren't induced in response to the hormones or the amount of induction or reduction in response to ovarian steroid hormones is different across the donors. Overall they still respond appropriately to estrogen based progesterone based on transcriptome studies but we know there are donor effects that actually might be interesting and useful to understand the mechanisms of steroid resistance. So recently we've taken the base media that doesn't contain protein components and we've done secretome analysis and so in this case what we will have looked have done through high throughput mass spectrometry is look for proteins that are in the media that would be secreted by the basolateral surface of the endometrial epithelial organoids and note that there are a number of proteins that are decreased versus the control and a number of proteins that are increased versus control. And what we have recently focused on are three proteins Cystatin C or CST3, Stanyl Calcium 1 or STC1 and Serine Protease Inhibitor A3 or Serpin A3. These are all secreted proteins that are increased by progesterone and are also differentially expressed in mid secretory versus proliferative phase endometrium. And what we saw in this experiment was to determine whether or not these proteins produced by the endometrial epithelial organoids and secreted out of the basal aspects could modify stromal cell decentralization given the proximity of the glands to the decentralizing stroma in a normal implantation site. So we use human endometrial fibroblasts. We grow them to 80% confluence and then reduce serum and do a typical decentralization cocktail protocol where we add estrogen and MPA and then either estrogen and MPA or that cocktail with PGE2. I'll note that PGE2 increases the expression of both prolactin and IGFBP1 over estrogen and MPA alone. And these are classic decentralization marker genes that are turned on when the cell is fully decentralized. Now addition of Cystatin C or CST3 at the highest dose reduced the induction of prolactin as well as IGFBP1 by estrogen, progesterone, and PGE2. So in fact, this was a little counterintuitive but we found that Cystatin C decreased decentralization but Serpin A3 and staniocalcin 1 had no impact on decentralization. And none of the three proteins had effects on stromal cells alone treated with estrogen and MPA. So this is some evidence that perhaps there are proteins or other secretive factors produced by the endometrial glands that can modulate stromal cell decentralization and we're continuing to explore that in our future studies. Now one of the consequences of this work or we know that defective decentralization has been implicated in the development or in pregnancy loss, placental insufficiency as well as preeclampsia. And so defects in uterine gland development or function may actually compromise the establishment of pregnancy and defects in uterine gland function may be involved in etiology of later pregnancy complications. And to be able to really successfully interrogate this what we need to do is be able to recapitulate the early human embryo or at least trophoblast endometrial interface and obviously it's ethically and really impossible to gain any insight because we can't get in specimens in vivo so we have to rely on in vitro techniques. So there's a couple of different approaches to do that and we are essentially using information published by other labs as well as trying to develop our own in vitro system. So this shows on the left some exciting work from Jan Brozens and Emma Lucas that were recently published in eLife where they've taken these asymboic cultures of gland organoids and stromal cells for implantation studies, pharmaceutical testing as well as you can use CRISPR gene editing to modify either the epithelium and or stroma. And so we're hoping that this type of approach will allow us to recapitulate early embryo maternal interactions. And we know that there's some other approaches and at Julie Kim's lab and others also have these type of asymboids and we're continuing to look and see what develops in terms of our own lab as well as others and being able to further refine these in vitro approaches and the use of organoids for the study of fertility as well as infertility and disease in women. Because as noted in this quite good review from Margarita Turcoy, this type of in vitro approach can be used to gain information on how do cells respond to bacterial infections, can we use the basis of organs on a chip, there's been some nice developments in that both at Northwestern as well as Vanderbilt and Linda Griffith's group at MIT. In addition, you can use a variety of other different approaches to really understand genetic modifications as well as drug screening for personalized medicine and ultimately we're hoping to develop biomarkers of normal and abnormal uterine function that are important for diagnosing infertility and pregnancy loss and also being able to develop stem cell therapy approaches for treating different types of diseases in women. So with that I thank you very much and again I apologize that I can't be there. Please feel free to reach out to me at the University of Missouri if you have any thoughts or ideas or would like to collaborate. Thanks and have a great day. So of course there's no formal Q&A for this but I figure just in case there's anyone in the audience here who just wants to make a comment or anything like that, feel free to do that. We have a few minutes. And if you do, please use the microphone. Also there's a QR code, I'm sure that we can email him the questions if you put your name on your comment as well, question. I think we can move ahead. Yeah, so I think at this stage we'll probably just go ahead. So our next speaker needs no introduction. It's Dr. Bruce Lessey who's going to talk to us all about epigenetics and genetics of endometriosis. Let's see. Advance. Oh yes. Great. Well, I appreciate the invitation and I would like to thank my collaborator Steve Young and Jaywook Jiang whose work together I will talk about today along with review of epigenetics which is a really big topic I came to understand. I have some disclosures including technology that's licensed and I'm on the advisory board for my event. So implantation as we heard is important and involves synchrony. A lot of this synchrony revolves around the ovary that produces estrogen. Estrogen increases the endometrial growth and readies it for implantation. With ovulation you have the embryo going down the fallopian tube and it's timed in such a way that the endometrium is ready for it when the embryo is ready to implant. All of this involves epigenetic changes and as I'll talk today, infertility can result from dysfunction in this process and this slide I borrowed from Tom Spencer shows the endometrium growing in response to estrogen on the left and it reaches a certain height and then the glands become secretory and the embryo is allowed to implant. On the right lower side you see decendralization which is critical for successful implantation and completely dependent on progesterone action. This is one of my favorite studies and came out in the New England Journal in 1999 by Wilcox at NIH, no it wasn't NIHS, it was across the road from you guys. It shows that most women implant during a window, the window occurs between eight to ten days after ovulation and in the blue bars are patients who miscarry and you can see that after day ten of the secretory phase the miscarriage rate goes progressively up. One of the conclusions of this study by the authors was that late implanting embryos or the embryo that was defective would implant late and that's why they had this, you could see this phenomenon. I think it's actually different, I think this is due to progesterone resistance and that patients who have delay in development of the endometrium force the embryo to implant late and move the window further away. When the window is later and the embryo is forced to implant later the corpus luteum can no longer be rescued and therefore the pregnancy is lost. We know this is probably true because if we give HCG shots around day eight or nine of the cycle we can rescue patients with unexplained recurrent loss to a tune of about 80% of the time. We obviously can't avoid euploid embryos, I mean aneuploid embryos from miscarrying but most euploid embryos will continue if you simply give a shot of HCG. This would suggest that the corpus luteum also plays a role in this phenomenon. I was told to talk about genetics and there are obviously genetic loci that have been discovered, associated with endometriosis and those references are shown on the left and some of these loci are shown on the right. In fact, no dysfunction has been discovered that relates directly to these genes in endometriosis. While this is interesting and may provide markers for population studies, it really doesn't inform us about what's going on, especially in the infertile patient who can't get pregnant. Cancer driver mutations have also been studied. They're listed here in the table, KRAS being one of them and I'll talk more about KRAS. I'll also talk about ARID1A in a minute. It's true that somatic mutations can be discovered in deep infiltrating endometriosis but cancer is extremely rare in endometriotic implants and so the biological significance of this is questionable. Further, the study in the New England Journal that talked about ARID1A as being missing because of mutations used immunohistochemistry as a surrogate for mutation. They didn't actually sequence ARID1A and it turns out that ARID1A is probably not mutated in most of these cases but epigenetically downregulated. So epigenetics is the study of changes in organisms caused by modification of gene expression rather than an alteration in the genetic code and involves histone modification, DNA methylation and mRNAs. It's a big topic and I won't cover it in any great detail today, unfortunately, but I will touch base on some of these mechanisms. Defects in endometrial receptivity arise primarily because of progesterone resistance. Tom Spencer informed us a lot of these. You heard already a lot of these markers being mentioned but it's been shown that PRB is downregulated along with chaperone proteins for progesterone receptor, implantation-specific biomarkers like COX-A10, the alpha-V beta-3 integrin and LIF are modified. The enzyme that breaks down estrogen is downregulated so more estrogen exists. SF1 and STAR and aromatase are all upregulated resulting in increased estrogen presence. There's inflammation through COX-2 and PGE-2 and inflammatory cytokines are expressed as part of the endometriotic phenotype. Associated with prostaglandins—I'm going to borrow this for a second—associated with prostaglandins is an elevation in MMPs associated with the endometrium of women with endometriosis. Virtually all of these changes that have been described have an epigenetic basis. I've been studying receptors for the estrogen and progesterone for I don't know how many years. I was at Colorado State University and worked on the dog uterus as part of my Ph.D. and studied ER and PR and then went on to look at it in human at Duke University. But a lot of the work that has come down comes out of Franco de Mayo's lab and shown on the right are some of the sophisticated pathways that they've discovered. But one of the primary objectives, the takeaway message I take from this is that one of the major reasons progesterone is there is to drive down estrogen action. You can see from this schematic that progesterone acting through Indian hedgehog and COOP-TF2 not only help decentralization, but they drive down estrogen receptors, which disappear at the time of implantation. If estrogen receptors don't disappear, as we're finding in our infertility patients in the IVF program, those patients are the ones who often have implantation failure. This paper from Adia in 2000 shows this phenomenon, that progesterone receptors are downregulated, at least PRB, primarily at the time of implantation in the human endometrium. But in endometriosis on the right, and this is specifically to the endometriotic stromal cells, PRB is completely missing. This may not be true for the eutopic endometrium. As we showed and Philippe Bouchard's group showed the same year in 1988, both estrogen and progesterone receptors are downregulated right at the time of embryo implantation. With progesterone resistance, this doesn't occur. This phenomenon of inflammation drives epigenetics. Inflammation is important and has a physiologic role in both implantation and menstruation. All these mechanisms that we hear are related to inflammation have a role during normal cyclic change. In this, you see that progesterone resistance, like progesterone withdrawal, both lead to progesterone to inflammation, and they're associated with multiple changes like MMPs, the COX-2, the inflammatory cytokines, and the others, all of which have been described during menstruation. Progesterone resistance is very much like progesterone withdrawal and probably causes the same inflammation through the same mechanism. Salmonson and Evans pointed out that if you give RU-486 in the mid-luteal phase, you can induce these same inflammatory changes. So I postulate that progesterone resistance is actually a physiologic phenomenon that's used during menstruation to help turn the endometrium around from a secretory to a proliferative structure in a matter of days. Without progesterone resistance, you might not be able to turn off progesterone, plus the inflammation that progesterone withdrawal incurs is important for the shedding of the endometrium. Unfortunately, for the endometriosis patient, this doesn't work out well for their fertility. So menstruation is an inflammatory event. It involves all of these compounds and the molecules that you see, and these are exactly the same molecules that you find in the endometrium of women with endometriosis. And epigenetics has a lot to do with these changes. DNA hypomethylation has been noted in SF1, aromatase, COX-2, estrogen receptor beta, PGE2, GATA6, VEGF, and hypermethylation is noted in those molecules I mentioned, like the enzyme that metabolizes estradiol, HOXA10, and even the alpha-B beta-3 integrin. Now I mentioned ARID1A. ARID1A is often talked about as a mutated gene. It is an inhibitor of inflammation. In this schematic on the left, you can see that ARID1A normally sits on a promoter that regulates inflammatory cytokine production, and when it's absent, it allows RELA to sit there, and this may be the reason why inflammation occurs in the setting of endometriosis when ARID1A is downregulated. ARID1A is targeted by microRNA, microRNA31, and therefore, this could be one of the major factors in starting the inflammatory process in endometriosis. This is nervous for me to be sitting next to Julie Kim and talk about signaling pathways, but progesterone enters the cell, binds to the receptor, which becomes phosphorylated and dimerizes, goes to the nucleus, and sits on various promoters and causes important gene expression, especially for decidualization, genes like prolactin, IGF1, BP1, lefty, etc. It also has a role in the cytoplasm interacting with Src and initiating AKT production. These pathways are very much interrupted in the setting of progesterone resistance. As we talked about earlier, Atiya had shown that PRB, for example, was downregulated, at least in the endometriotic stromal cells, suggesting that progesterone resistance was really a lack of progesterone receptors, but I think it goes actually deeper than that. Inflammation in the form of these cytokines has many effects on the endometrium, including the development of COX-2 and prostaglandins. PEG2 initiates the production of SF1, which turns on the entire steroidogenic pathway, which inappropriately makes estrogen in the endometrium through aromatase. In addition, progesterone produces CUPDF2 and WT1, which interact to block NF-kappa-B, which is an inflammatory marker. In progesterone resistance, this does not occur. We recently showed that the T cells that make up the endometrium, 40% of the endometrium is involved with leukocytes, are usually Treg cells, but the ratio changes, as shown in panel C. When the ratio changes and you get more Th17 cells than Treg cells, those are the patients who have implantation failure in our IVF program. Now Serdar Bulan has discovered epigenetic switches, including GAT2 to GAT4 and GAT6, and again, this is in endometriotic stromal cells, but I'm not sure this applies to eutopic endometrium. I've never seen data that shows this in the normal or abnormal endometrium. As you can see, GAT2 is responsible for many of the proteins that I talked about coming out of the DeMeo lab, and GAT6, when it switches to GAT6, is involved in downregulation of those genes and upregulation in other things associated with endometriosis. This is a good review, which I bring up to share with you, talking about some of the histone deacetylases in SIRT1, and I'm going to finish the talk today talking about my favorite molecule, SIRT1. In our current model, inflammation leads to elevation in KRAS. KRAS is primarily responsible for stimulating SIRT1. SIRT1 interacts with the progesterone receptor, along with BCL6, and acts as a transcription repressor, turning off many of the genes that are essential for implantation, including GLE1 and FOXO1. In the meantime, KRAS is stimulating estrogen signaling, so you get an estrogen-dominant state, and in fact, estrogen receptors do not downregulate in women with progesterone resistance, as expected, and that leads to more proliferation, invasion, and survival. We published in, I think, 2000, or this is 2017, this U paper showing that KRAS and SIRT1 were co-expressed and elevated in the endometrium of women with endometriosis. We also know from our animal models, from Jaywook Jeong and Azgi Fazlibas, that like the human with endometriosis, where SIRT1 is elevated, in the baboon, if you induce endometriosis, SIRT1 goes up by nine months and reaches a peak by 15 months, suggesting that it takes time for these inflammatory changes to be set up and for progesterone resistance to be maintained. The same thing happens in the mouse. If you over-express SIRT1, I mean, if you induce endometriosis in the mouse, SIRT1 is upregulated, as shown in panel C. SIRT1 has implications for survival. It is a survival gene, and in fact, when we over-express SIRT1, as Jaywook Jeong has done in his lab, using a live imaging model of endometriosis, shown in green, you can see that endometriosis worsens when SIRT1 is over-expressed. Not unexpected, perhaps, because of its survival implications. In women, as we've just published in JCEM, normal women only express SIRT1 during the menstrual phase of their menstrual cycle, at the time of inflammation, but in women with endometriosis, SIRT1 is elevated throughout the menstrual cycle. There's a very tight regulatory control loop associated with SIRT1 that involves microRNA 34A and P53. SIRT1 deacetylates and inactivates P53, and P53 is responsible for upregulating microRNA 34A. 34A, in turn, downregulates SIRT1. As shown recently in this very nice paper by Risk et al., there's an inverse relationship between SIRT1 expression and the microRNA 34. They also provide evidence that FOXO1 is directly inhibited by elevated SIRT1. I've shown this slide earlier, and it just points out that this is a survival gene and important for the pathophysiology, we think, of endometriosis. In mice, where we over-express SIRT1, Jaywook has shown that implantation goes away. These mouths become profoundly infertile, as shown in B. They also have decidualization defects, as shown in C, and overactive proliferation, as shown in D. If we give an inhibitor of SIRT1, EX527, to these mice, we can rescue the implantation defect, and these infertile mice then become fertile, again suggesting that SIRT1 is at the center, perhaps, of these epigenetic changes and the progesterone resistance that follows. Finally, BCL6, the pairing partner with SIRT1 is elevated as well. It is probably regulated by IL-17, and inflammatory cytokines. It co-localizes with SIRT1, as shown in this slide. And in a recent review, talking about preeclampsia and endometriosis, they provide evidence that BCL6 is probably involved in stemness, stromal cells, and the immune response, and proliferation and differentiation of the endometrium. When it's elevated, these become dysfunctional. So treatment targets abound in the epigenetic field. As I finish up, we can inhibit COX-2, as we've often done for pelvic pain, associated with endometriosis. We can suppress menses and estrogen with Lupron and other things. Or we can remove surgery to remove, we can use surgery to remove the lesions. And certainly there have been papers showing that when you use Lupron for two months, patients have a higher implantation rate than when you don't. We used BCL6 to study IVF and showed that high BCL6 patients had a lower implantation rate. And when we treated them, as shown in blue, with the GnRH, or orange, with surgery, we could improve the implantation rate compared to the untreated controls. Note that the miscarriage rate is also much higher in the untreated controls, compared to those who received treatment. So in summary, endometriosis is a terrible disease. It causes misery around the world. Costs well over 22 billion per year. Infertility and pelvic pain. Epigenetics plays a major role in progesterone resistance and steroid hormone dysfunction. And inflammation, ultimately, we think, causes many of these epigenetic changes and induces a menstrual phenotype that is incompatible with pregnancy. Thank you for your attention. Do we have any questions? Dr. Taylor, Buffalo. Thanks, Bruce. That was really nice. Great review. And I mean, this is a wonderful area, obviously, to be studying right now. Do you think that there's a direct effect of inflammation and the sort of cascade that you showed on progesterone resistance or progesterone withdrawal or some of the other phenomena? Or do you think that it's conceivably mediated through some of these micro RNAs that you've kind of described in the SIRT system? I'm kind of curious what your thinking is about how the progesterone resistance comes about and if that's directly linked to inflammation. It's a really good question. I still ponder the chicken and the egg. How do you get to the point of inflammation without some of the micro RNAs that have to downregulate ARID1A, for example? So I think the inflammation probably arises from the presence of foreign endometrium, or endometrium that's not supposed to be in the pelvis. The immune system reacts to it and that starts the cascade. And again, it takes a while. In the baboon, it takes nine months for this to become established, apparently. Any marker you look at, it takes about nine months before it happens. So I think it takes time, but it's involving probably ectopic endometrium. So, Alfred DiMaio, NIEHS, so very nice talk, Bruce. So there's this battle going on between progesterone receptor and FOXO1. And I think, as SIRT regulates PR and stuff, so how important do you think that that battle is in endometriosis? The uncontrolled FOXO1 when progesterone receptor goes down and stuff. And it's all through AKT really modulating that. So can you comment on that? Well, FOXO1 is important for decidualization. So that's a conundrum, right? Because I think we and others have shown that FOXO1 regulates PR's ability to turn on genes, but PR keeps FOXO1 out of the nucleus because if it's too much in, you get apoptosis. So there's some tider between PR levels and FOXO1 to keep just enough in the nucleus to allow it, but to keep it from causing apoptosis. I mean, that's a real interesting- Well, this may be one of the reasons why progesterone receptors need to disappear from the epithelium at the time of implantation. They've already initiated FOXO1 expression, and then they need to turn off. And so implantation is really a time of withdrawal of progesterone support in sort of a paradoxical way. Yeah, I mean, there's a battle going on because if you push PR, FOXO1 doesn't go away. If you knock out PR, FOXO1 stays. It's a really interesting question. Yeah, thank you. I have a question. So SIRT1 is a deacetylase. It is, histone deacetylase. So have you looked at what it deacetylase? I think it deacetylases histones as well as other non-histone proteins. Do you know what's going on in your system? Jaywook Jeong has looked at a number of genes, and I presented that in one of my slides, but didn't point it out on the right-hand side. We know GLE1 and FOXO1 are two that it interferes with. COOPTF2, an important mediator, might be regulated that way. But we need to do those studies. And I have another question that might be a little harder to answer. So I don't think the number has changed in terms of the incidence of women that get endometriosis around 20%. I don't know if that changes, but that's what I've seen in the literature over the past many years. Yet a lot of the animal models that we induce endometriosis get endometriosis almost 100% of the time. We know that inflammation is involved, and I'm assuming it's because there are lesions outside of the uterus, and the immune system is just kicking up a notch there. So what do you think is actually allowing these lesions to form in endometriosis, even before there is inflammation, or is there already inflammation in the eutopic endometrium? I'm asking this question because I think there is a huge genetic component that we still don't know, that we could potentially look at to see what causes the establishment, and I would love your insights. Yeah, I could talk all day on this topic. First of all, there are a lot of women with endometriosis who don't know they have it. We're not doing laparoscopy anymore, basically. The attorney said laparoscopy is dead. The only thing left is the obituary. We do IVF, and so the women who fail IVF in our program with euploid embryos, 80% of them have endometriosis, we're finding, or at least markers of endometriosis. When we treat them with Lupron, we have a 90% pregnancy rate with euploid embryos. It's pretty clear that if we don't treat them, they have a 37% pregnancy rate. There's endometriosis out there, and we don't know it's out there, so I'm not sure if that confuses the situation or clarifies it. Thank you. Another question. So, our final speaker this morning is Dr. Carlos Simon, University of Valencia in Spain. I don't have Dr. Simon's presentation title in front of me right now. Okay, here it is. So, Single-Cell Transpectomic Atlas of the Human Endometrium and Health and Disease. And I'm sure Dr. Simon is going to tell us about some exciting work that was recently published. And if you haven't had a chance, there was a paper published in Nature Medicine about a year or two years ago, and I really encourage everyone to go and look at that study as well. Thanks very much. Thank you. Thank you. You almost spoiled my presentation, I tell you. Well, thank you for the invitation to be here for me. It's a pleasure and an honor to be here with these two greatest speakers and to be able to inform you about what we are doing on the human endometrium. First, sorry, I will just have the disclosure that you have here. And basically, the point, the overview of my presentation is going to be to understand a new vision of the human endometrium at the single-cell resolution, and then to learn about what this technology can offer to advance our basic knowledge of endometrial pathologies. And for that, I will just touch upon endometriosis, atrophic endometrium, and Asherman syndrome. Well, I think that you know everything about the endometrium that I can tell you. This is a very focused meeting. And this is a very interesting organ that we are all in love with. And that's why we have devoted our life, the oldest in this room, to understand this. What I can tell you basically is that the function, this is a reproductive immortal organ. And the main function of this endometrium is to open a window of implantation, allowing the embryo to adhere and to implant. And that's why cyclicity comes and all the things that comes with that. And I mean, since a long time ago, I was really falling in love with how this can be true and how it happens. And the fact that I'm going to tell you about single cells is another step forward in the understanding of this remarkable organ and this remarkable process. So we have seen the human endometrium with our bare eyes, with hysteroscopies, and we have done diagnosis of everything. And this is what we do every day. And we remove any myoma or any type of pathology with that, with our bare eyes. Then it comes histology, amazing histology, and the most quoted paper in obstetrics and gynecology. Since this seminal paper in more than 70 years ago, Noyes and colleagues, they really put forward dating of endometrial biopsy. And this is very interesting. And since I was in Boston, I learned what was the real history of how they got these eight major histologic criteria. And the way that they did it at that time was, as you can see here, they reviewed 300 protocols of patients, 40 of them, only 40 of them, they were correlated with basal body temperature at that time. And they did, 13 of them, they photograph. And out of these 13 patients, they got all of these eight major histologic criteria. Amazing, they have been following for more than 70 years, and we have based all of our knowledge on that. Knowing that today, they will not resist any peer review exercise. But this is what we are coming from. Next has been imaging. Everything is anatomical medicine. So imaging has been great. In fact, this is what we are using nowadays. And we do not conceive our clinical practice without vaginal ultrasound. We always mention about thickness and pattern. And we know how to categorize this. Some people are obsessed about seven millimeters, eight millimeters. We know that at the end of the day, this is illustrative, and you can see an atrophic endometrium, which is bad news, or you can see a hyperplastic endometrium, which are also bad news. But we are aware that they cannot be used, really, to understand receptivity or healthy endometrium at all, in general terms. And there are many publications with many statistics about that. And then, about 10 years, about 20 years ago, excuse me, it came molecular medicine in the scenario, by which we can really objectively try to understand this organ from a very molecular point of view. And these are the pioneers' work from Bruce Lacy, from Linda Yudis, Peter Rogers, for ourselves, from 2003, by which we can really recognize endometrium and the endometrial biology, and all the different cell types throughout the menstrual cycle. And from that, we just publish and put forward what is the transcriptomic signature of endometrial receptivity that now is being used clinically worldwide. Now, but this is bulk tissue. This is the whole tissue, and this is just the transcriptomics. And this is an objective criteria, but either way, this is a bulk tissue. And as you know, our tissue is quite complex. And our tissue that we are discussing here has not only different cell types, but also those are in a different cell cycle, and we have also the timing of the menstrual cycle that we are living with. So, it will require, this requires that we go deep in the understanding and go for the next technical advances for that. And this is why single-cell RNA-seq... Excuse me. This is why RNA-seq came to the room here. Single-cell RNA-seq is to the actual technology as Google Map is like a map. We used to go to places using a map. Now, Google Maps let us know exactly what is the place, what we should go. So, this is the idea of the Human Cell Atlas. The Human Cell Atlas has transformed the way that we understand anatomy worldwide. And you know that this is a human cell. And you know that this is a huge consortium worldwide, more than 2,000 members, more than 1,000 institutes, 83 countries. You can see the founders, Sanger Institute. You can see it worldwide. And those are the working groups, and they are just trying to understand each organ and having now a different viewpoint at the single-cell level. And of course, reproductive organs are not the exception. And this is what I'm going to tell you about. Focusing, of course, in the uterus. The technology is quite straightforward. There has been different advances from fluidism to actually 10X that allow us really to have this understanding, obtaining the cells, separate the cells. And then individual cells, they get bar-coded with beads isolated in oil droplets. And then they can go directly to reverse transcription, library generation, and finally high-throughput and having the transcriptomes of single cells of these 30,000, 40,000 genes transcribed at the very same moment. So the possibilities are endless. And this is just cells, but now it comes, as we discussed with Franco De Maggio this morning, spatial transcriptomics, that they are not only giving you what are the cells there, but also where they should be located. I think both things, they are quite powerful that at the end it will come with proteomics and so on. So science cannot stop. So we should really go in the winds of these technical advantages. So I would like to mention what we published two years ago about the single-cell transcriptomic atlas of the human endometrium during the menstrual cycle, setting the pace for what is the healthy endometrium, what is the contra-endometrium, and then to have this as an initial point to go to pathologies. In this paper that we published in collaboration with our colleagues at Stanford, Steve Quake Group, that is very well-known, is at the Biohub, and he's also participated on the human cell atlas. In this paper, basically we analyze more than 70,000 cells from 29 healthy ovum donors throughout their menstrual cycle. We obtain their biopsies, we separate them, and at the end we go to the single-cell isolation, cells that they go through different steps, and once the cell is combined in the oil drop that I mentioned to you, then we have the analysis. We start with fluid digging, because this work was started four or five years ago, but at the end, after the request of the reviewers, we have to double-check with 10X, and we went through the whole process. Now, what we learned from here was amazing. First of all, we can really differentiate the different cell types of the human endometrium just by having the transcriptomic signature. It's quite obvious, but for us it was very important to learn these six cell types. That they can be differentiated, that if you just take endometrial fluid and you just pick up one cell, you know where this cell come from by having this identity card, transcriptomic identity card. So from this, we learn, of course, that there is epithelium, but the epithelium, also we discovered that there was a ciliated epithelium, something that has been already described, but there was rare cells there. So we have what we call unciliated ciliated epithelium. We have, of course, the stroma, but we have also the immune cells that we can distinguish between macrophage and lymphocytes, and then we have also endothelial cells there. So these are basic six cell types. As you go further, as you will see in the next papers, we can really go to 14 cell types, but I think those are the basic cell types that you can get started with. This is static. Now, if we go specifically to the secretory phase, now you can see those clouds of cells that, this is epithelium, for instance. You can distinguish between luminal and glandular epithelium, and I think this is for, thank you, for Thomas Spencer work, I think it will be, because it has been puzzling always for us how we can distinguish epithelium, glandular from luminal epithelium. So now, we can do really easily here, and as you can see, we can distinguish also the ciliated epithelium, the stromal fibroblasts, endothelium, lymphocytes, and macrophages. Within this technology, we have in one spot the genes here that they are expressed and how the expression is in each of these different cell types. Very important, we learn about using this pseudotime construction, which basically, we use this method based on mutual information. We try to learn how the timing is presented within the menstrual cycle. Typically, we always understand that we have menstruation, we have proliferative phase, regenerative, proliferative, then we have the secretory phase, and we have the window of implantation, and then things get started again. We understand that this is the logical way and the logical transcriptomic of the biology of the endometrium, and this is true for the stromal compartment. So, as you can see here, one, two, three, four is how they just develop in terms of transcriptomic modifications, and the big gap, the discontinuity, goes from the late secretory phase to the menstruation, which is quite logical. However, look at the epithelium. In the epithelium, this is not the case. The big transcriptomic gap is present between phase three and phase four. In other words, it's in the opening of the window of implantation. So, somehow, the big transcriptomic differences arises in the epithelium when the window of implantation is open, and I think this is remarkable. And you can see this now in this graph, in which, in the upper part, you can see, we call it ciliated epithelium to distinguish from the ciliated epithelium. This is the normal ones. You can see how this is developing throughout the menstrual cycle, and just here, there is an abrupt change of acquisition of turning on those genes, and this is just the opening of the window of implantation. This is different from what happened in the stromal compartment, in which this decidualization changes are more subtle, and they are being just modified without these big jumps in terms of transcriptomic opening. Also, we learn, and this will be very interesting for all of you, but also for Thomas Spencer, that we are ready to understand how the epithelium can go from the glandular to the luminal to understand those differences that we have now in each of those cell types. And even more, we have learned how the luminal epithelia, how it is producing the glands. So, gland formation is also getting some initial hits from this type of technology, and how the glands will develop from the luminal epithelium. And of course, the lymphocyte stromal interaction during this individualization is key. Those cells, and you can see in the figure of the papers that we did the morphology, how these cells, they just touch each other, they can communicate, and the language that they use in terms of using NK receptors and using specific interleukin as interleukin 2 and interleukin 15. As we discussed with Kim also before, more and more data they are just sending us through immunological pathways, which are really difficult to understand, but the immunology is there, and we should pay attention to that. Now, in conclusion from this part, and this means to be just the healthy controls, the healthy endometrium, what we learn is that our unbiased single-cell analysis allows for definition of global feature of transcriptomic dynamics as a step forward to classical histology and whole tissue transcriptomics. We identify six major endometrial cell types and four major phases of endometrial transformation regarding the epithelium is different from the stroma. We describe also differences between luminal and glandular epithelium, and we just describe again this abrupt transcriptomic opening of the window of implantation at single-cell level, somehow reinforcing what we already described in the bulk tissue, in the bulk endometrium. The sedolization, interestingly, we always used to say that first is the window of implantation and then comes the sedolization, but it looks like it's initiated even before the opening of the window of implantation, and is widespread in a stromal fibroblast during this window of implantation. And, of course, the direct interplay between the stromal fibroblast and lymphocytes during the sedolization in natural cycles. So, this is just, this is the baseline that we think is what happens in the, let's call, healthy endometrium. They were coming from donors, that they were fertile. And now, what happens, what is about the endometrial pathologies? So, the field is starting at the single-cell level, of course, and there are few papers. And, in fact, I'm going to show some of them to you. This paper is the only one that I have found in endometriosis, and this was at the time that this is in the preprint, in bioarchives. I know that this is under editorial consideration, and probably is in a very good direction. So, that's why I show you this here, but I cannot enter in details because of the time, and also because has not been peer-reviewed published yet. But, in this particular paper, the authors, they got to a very interesting, very interesting conclusions that I try to summarize here. First, the number of cells analyzed and the individuals were just fine. And, when you read the paper, the most important message is that they revealed the existence of an immunotolerant peritoneal niche, which is something really interesting, that now the interest is moving to the niche that somehow accepts the fact that those cells, they engraft there. And, they also discover, of course, fundamental differences in eutopic endometrium and between lesions microenvironments and a novel progenitor-like epithelial cell subpopulation. So, this is the message that I just got from this paper, which, of course, you can read it, and I think this is refreshing to give us another view that is not only the cell, but also the niche that they are going to engraft, maybe induced by the difference of those cells, but maybe not. But, this is the only thing that I have found of endometriosis. Maybe you have other information, but this is what we have. And, the other pathologies are the endometrial stem, what we call endometrial stem cell niche, which basically is when you have the niche that is broken and they provoke either artificially the replacement of bona fide endometrium by fibrotic tissue. This is clear for Asherman syndrome, and endometrial atrophy somehow has been suggested that this may be due to the absence of those endometrial stem cells that, at the end, they will not be able to produce a proper endometrium. Regarding to the thin endometrium, there is one very nice paper published, which is this one in PNAS, just recently, in which, basically, the number and patients, the number of cells and patients, they were just very, very acceptable. And, the conclusions that they obtained from this, it was that there is a cellular senescence in the stroma and epithelium accompanied by collagen over the position around blood vessels. You can see here how they started. This is the normal endometrium, this is the atrophic endometrium, this is the plot that I show you, and these are the graphs indicating this. They discovered, they found a decreased number of macrophages and NK cells, and they discovered an aberrant pathways that you have here, CMA3, EGF, PTN, and TWIK, as causes of this insufficient proliferation of the endometrium. So, very interesting paper to read, and they can give us some clues to those that they will be working on the atrophy. And, finally, I want to show you our unpublished work on Asherman syndrome. It's unpublished, so I bring it here just for you because I think that this is a high-level meeting, and I would like just to show you what we have. As you know, Asherman syndrome is defined as the presence of intrauterine adhesion causing the uterine walls to adhere to one another, resulting in the clinical symptoms that we know. But, unfortunately, this is the very same definition of Joseph Asherman almost 70 years ago. And the problem of this definition that they described for the first time, this traumatic amenorrhea. As you know, this is provoked by curettages specifically or more frequently in the postpartum. The problem of this definition is that when we see this, and this is, we take this definition, the only cure that we have is to cut the adhesion, and this is what we do gynecologists all over the world. To go there, to remove adhesions, and to put some barrier, and then we go home. And with that, maybe we will solve half of the cases, the most easy cases. But we know that in the majority of cases, 50% of them, they are refractory endometrium there, and the refractory Asherman syndrome, and those patients will not be able to conceive. So, using this technology, what we have done is taking the advantage that we were having in one part, one phase, two clinical trials authorized by the European Medical Agency. And on the other part, we have funding for European Union as the project called UTER that I will introduce to you. We have analyzed this pathology in depth. We study more than 110,000 transcriptomes from 10 patients with moderate severe Asherman syndrome, and compare with these 68,000 transcriptomes from healthy ovum donors. As you see here, I know it's difficult to see that, but basically these are just Asherman syndrome, and these are healthy patients. What you can see, for instance, is the epithelium of the window of implantation is almost disappearing here, and I will show you the difference in the different cell types. But we discovered three clusters which are different from Asherman syndrome to healthy endometrium, and those cluster has to do with mite cells, those are specific regulatory T-cells, has to do with immune-related epithelial cells, and has to do with specific stromal cells, that they are unique in the Asherman syndrome. When we compare the different cell types, as you see here, this is first Asherman, and then is control, you can see that the epithelium is very much reduced in Asherman versus control, that the dendritic cells, they're highly present in Asherman and almost non-in controls. The epithelium of the window of implantation is almost non-existing here, and so lymphocytes are similar, endotheliums are similar, perivascular are okay, macrophages are highly expressed here, they are increased, so we can see differences. And moreover, when we analyze the different cell types, even if they are just categorized as such, the genetic expression of the epithelium is different when we compare Asherman syndrome versus when we compare a contra-endometrium. Look at our molecules as a glycodelin and so on, that they are really low, very low expressed in the Asherman syndrome compared to the normal endometrium. So even in the different cell types, the expression is different. Now, to understand to what extent this is important or not, we use the model that we have. As I said, we are in phase two of this clinical trial with CD133 bone marrow derived stem cells. There are autologous cells that we introduce to the patients via uterine artery, and this is the profile of the bone marrow stem cells that we are using. And by doing so, what we have learned is that, again, we modify back the profile of these cell types, you can see it here, and when we compare now the cell ratios that we have after the treatment, you can see that the dendritic cells, we do not move the dendritic cells, we improve somehow the epithelium here. The stroma is the same, macrophages are somehow, they are just changing, but they are not getting to the normal situation, but we use this as a counterpart to understand at what extent we get closer to the normal situation. And so is the same with the specific marker genes, a specific transcriptomic signature in each cell types that you have here. So from this work still set and published, what we learn is that there is a differential transcriptomic and cellular cartography of the human endometrium in Asherman syndrome that reveal that there is alterations in the menstrual cycle, tissue remodeling and angiogenesis. The most important thing is that Asherman syndrome is characterized by disruption of angiogenesis, this is what we learn in our genetic profile, by an inflammatory response which is leaded by macrophages and T-cells and fibrosis. So anti-angiogenesis, pro-inflammatory and fibrosis. And the use of this system that we are trying to put forward indicates that we can just reduce this gap existing between Asherman and normal endometrium. So somehow the point that I want to make is always the same. If we are just, if we just point to the moon, which in this case is exactly to know what happens, don't look at the finger, because now we are retaining what is the problem of the fibrosis, and if we just keep cutting additions and not going to the place where the action is, we will never be able to solve this pathology. Sorry, I just want to emphasize that this work has been possible because we have this European project which is called HUTR, the Human Uterus Cell Atlas. This is founded by Horizon 2020 and it is composed by six international partners and this is the founding that we have. And the purpose is to create a single cell and a spatial reference map of the human uterus trying to move from the healthy endometrium to the endometrial pathologies as you have seen in this presentation. These are the partners that we are working with them, the Sanger Institute, Uppsala University, University of East Anglia and an Estonia group. And thank you very much for your attention. So very nice, extremely nice. I have a specific question and a bigger picture question. So the ciliated loss, the gap in the epithelium, does that just indicate estrogen's action? Because it's been shown by Turco that estrogen is regulating ciliagenesis in the organoids and stuff and it would fit that. Where estrogen drops at the mid-secretory phase, it goes down and then when you get near the end, it comes back. So do you think that's the driver of the ciliagenesis and you're seeing in your epithelium through the cycle? Probably yes, you are completely right. But what we have seen is that there are interactions with the immune molecules that maybe also will interact to that, maybe blocking the action of the estradiol receptor. So I don't know whether it will be a straightforward effect or just mediated through this cloud. So that leads into my, you led right into my comment. So genomics is great, it's highly specific and it's really good, but don't you think the future is going to be improved proteomics? Because there's a lot of things that you don't change at the transcriptome level, but the protein activity changes. I mean, we found several molecules, not by looking at microarrays or single cell, but by doing kinomassase or RIME or some pull-downs that really tell us what's going on. In fact, they're the druggable targets that you wanted to do the treatment. So do you think that the future is really going to be improved proteomics at the single cell level or the spatial level? Absolutely. And this is happening as we speak. But you know that this has been taking so long. I mean, your conclusion is so clear and we can agree with that. But we are waiting now for that long that proteomics will be up to the level, not up to the level of single cell, but up to the level of transcriptomic for obvious reasons. I mean, protein, you know that this is a 3D, I mean, we can talk about that. But yes, fully agree with you. We are collaborating with some colleagues at the Max Planck Institute that they have developed this single cell proteomics, but it still is not there. You know, it is, yeah, certainly it will be there, no doubt about that. But still, transcriptomics is our best shot. Even if it is not that direct, you have, you obviously, this post-transcriptomic formation, but yeah, fully agree. Carlos, beautiful work. The ATLAS and the single cell transcriptomes that you've described are amazing. We really need to move into pregnancy, however. Do you think that this single cell technology is going to look at early pregnancy and early pregnancy loss? Yeah, I think so. I think that this is going to help us to understand many things from non-invasive. I mean, we can search for that now in the endometrial fluid, and we can search for that in a specific situation. I mean, the Sanger Institute, they published this at the very early implantation sites, learning about the different, there are six types of decidual cells, depending on how deep you can found it close to the myometrium. So, I think that this is going to help us a lot to really understand what is going on. And then, could you elaborate on therapeutic ideas that you've come up with, with the Asherman's data? Yeah, basically, it's in line with your line of thinking. I mean, this is not about to go doing surgery. If we can really pinpoint these three main effects, anti-angiogenic, pro-inflammatory, and pro-fibrotic, maybe we can go with drug world targets that we can use to the patient instead of operating them again and again, just to avoid that it will happen. We don't have targets yet. We are just at this stage. But I think this should be the way through because, as in endometriosis, we cannot keep operating patients and removing endometriosis implants and then having them back. I agree. And same for myomas. I mean, this is where we have to try to be better than we have done. Beautiful work. Thank you. Hi, I'm Margarita Pisarska from Cedars-Sinai. Really beautiful work. Did you look for the Asherman's patients and even looking at the signatures because it looks like there's differences in some of the cell types between the Asherman's and the controls. Did you think about looking or have you already looked at upstream regulators of those signatures just to even try to get an idea of might there be, what are the transcriptional regulators or are there targetable things that could be regulating those differences in the gene expressions of the cell types? Absolutely. I mean, we are trying to explore now what is the situation. We are really amazing about mite cells. Maybe you have heard about that. Those are T regulatory cells. I tried to get away from immunology, but in those cases, it's so obvious that you can go there. And if you try to make the link clinically, if you see Asherman's is produced in the main majority of cases, 99% when you do a curettage in the postpartum. I mean, postpartum, it's a moment that there is a immunological situation there and maybe you are inducing this that gets perpetuated and then from that you produce this fibrosis all over there. But these are just hypotheses. Yes, of course, we are trying to do a 360 after these results to understand more and see what could we target in those patients. Thank you, Carlos. I was just going to make the comment that I think you kind of understated some of the changes, particularly in the macrophages, I thought, in your stem cell treated population. So those numbers, they didn't completely normalize, but they came down quite a bit from the untreated Asherman's syndrome. I'm kind of curious, are there subtypes of macrophages that you think that might sort of explain some of these things? And have you, you know, sort of been able to look at, you know, type 1 and type 2 and now all the whole range of different? Yes, absolutely. You get it in the, just like that. Yes, there are different subtypes. In fact, with this study, we are getting now, we are narrowing to 14 cell types instead of 6 initially. But doing this by having a specific subpopulations of the different T cells, macrophages, and this is what is getting interesting. I don't want to go too deep because then we will get lost. But yeah, those differences are really interesting and we are trying to make sense of it. I have a question. With my limited knowledge of immunology, and you're right, all of our omics data is pointing towards immunology, I know that NK cells that are in the uterus are very phenotypically different from those in the bloodstream. So how do those immune cells get in the uterus? Are they resident immune cells or are they influxed from the circulation? This is a very old and difficult question. What is clear is that they are different. Whatever you do in blood doesn't have anything to do with what is there. Whether they go there and they have this or they are just evolved from the tissue remains to be known. But we always try to be local. I mean, we try to go to the place where the action is, not go to the blood and then to extrapolate what happens in the uterus. So I cannot answer specifically to your question, but the point is to go for what is happening in the endometrium. So those NK cells, they were just there and we were able to really locate them and find them close to the stromal cells and find how they communicate. And you can see in the pictures of the figures of the paper. Great. So please join me in thanking all the speakers for an absolutely fantastic session this morning.

endometrium

epigenetics

endometriosis

organoids

infertility

progesterone resistance

inflammation

epigenetic changes

SIRT1

progesterone receptors

BCL6

steroid hormone dysfunction

genetic component