International Medical Conference Endometriosis 2025:
Endometriosis 2025: Your Mother Should Know, Your Doctor Should Know Better!
Single Cell Transcriptomics in Microenvironment of Endometriosis Lesions - Danielle Luciano, MD
So it's my pleasure to introduce our next speaker, who is Dr. Danielle Luciano. She is a professor of Obstetrics and gynecology and a fellow trained in minimally invasive gynecology at Yukon Health. She serves as a director of the Center of Excellence in minimally invasive gynecologic surgery and the fellowship in minimally invasive gynecologic surgery at uc Health. And she also is a co-founder of the Endo Rise project that is a state run project to collect samples for endometriosis from endometriosis patients welcome. Thank you.
Thank you. Thank you to Dr. Kin and Dr. Martin for having me here today. Thank you for the Endometriosis Foundation and all it does for endometriosis patients and for also supporting what we do over in Connecticut and Dr. Martin for supporting our Endo Rise program as he is part of that as well. So the Endo Rise program is my only disclosure. I am the co-director of the Endo Rise program and it is state funded. So this is our program. It is a program dedicated to both the collection of endometriosis data and tissues for our bio repository, which we are proudly part of the world Endometriosis Research Foundation, biorepository as well, using their SOPs.
But it also is dedicated to education of both patients and providers so that we can all, as I say, treat, manage, and help patients with endometriosis as a team sport. I really think that's what that's about and that's why I love being at programs like this. It shows all the team members. So the objectives of my talk today are to review the technique of single cell transcriptomics, which at least half of way more about than I do, and how it can be used to understand endometriosis, review some of the current literature looking at single cell transcriptomics, and discuss the importance of collaboration between clinicians and basic sciences for the future of endometriosis research. So what do we know? We know what endometriosis looks like. We know that endometriosis causes pain and infertility and all sorts of other things, but we don't know exactly how endometriosis causes pain and infertility and why it causes pain in some and infertility in others and vice versa. We don't know what's happening at the cellular level in these lesions that we excise. And what do we need? We need early noninvasive diagnostic testing. We need targeted personalized therapies to cure endometriosis, and we need ways to improve fertility in endometriosis patients struggling with infertility.
So in our lab, Dr. COIs is the director of the single Cell Biology Lab, and she is my co-partner and co-founder of the CT Endo Rise program. We're doing cutting edge single cell technologies. I usually use a food analogy. I have an Italian background and food works really well with me, but we also use Legos. So just for those physicians who don't know all of the different stuff, this is bulk RNA, so all of our Legos all mixed up. Single cell assays take our Legos and put them in little groups. And then spatial omic tells us where the little groups fall. And then functional tissue is a 3D model of that. So again, like food, but Legos this time. So Dr. Tan, who is here today as well, we did a study looking at single cell analysis of endometriosis, and we revealed some transcriptome program driving immuno tolerance in angiogenesis across eutopic and ectopic tissues. And so, oops, let's see. There we go. So what did we do first? First, we identified all the cells forming the complex microenvironment of the lesions and the endometrium. So we took the bulk RN aeq, the big pile of Legos, and we made 'em into small groups of Legos. So here are our endothelial cells, here are our stromal cells, here's our lymphocytes, epithelial cells, and myeloid cells. So this tells us what kind of cells and how many of those kinds of cells exist in our pile of legos.
So when we did that, we found lots of differences. We looked at differences between the utopic endometrium of endometriosis patients, and we looked at between those and controls, and I forgot to mention this, all of our patients in our study were on progestin therapy. This was a pilot study. And so we were trying to, didn't think we'd be able to get enough controls and endometriosis patients in all the various moments of the cycle. And we're going to talk about how these cells changed during the menstrual cycle. And so we got them all in on progestin therapy so that they would all be essentially in the same phase of their cycle.
And our control patients, by the way, were, this is always a discussion. What is a control patient? These were patients who did not have any pain and patients who were having a tubal ligation and not all of the patients had had children. So I suppose I cannot say that there was not infertility. However, all of these patients were having a tubal ligation, so they did have a laparoscopy that confirmed no endometriosis. Oops, how do I go back? Okay, so what we saw was that the epithelial cells, so the olive green make up the bulk of the tissue in the control endometrium and the fibroblast, which is the teal, make up the bulk of the endometriosis in endometriosis endometrium. And we also saw that the immune cells differ with higher proportions of immune cells to epithelial cells and more T lymphocytes, which are the red and more NK cells, which are the dark green in the atopic endometrium of endometriosis patients versus the atopic endometrium of control patients.
We also looked at endometriomas and endometriosis lesions. So epithelial cells are, again, olive green. Here are very minor components of the endometrioid lesions and stromal fibroblasts and endothelial cells make up greater than 70% of the lesion. We also saw that myeloid cells, the light green and endothelial components, the pink in ectopic lesions are almost twice that compared to eutopic endometrium and the adjacent peritoneum, which is really interesting. The adjacent peritoneum still has components consistent with endometriosis. So the question is, what is the margin that we need for these diseases, right? So I'd like to think that I'm a pretty good endometriosis surgeon and I try to get really good margins and get good healthy peritoneum around my lesion, yet I still see a pattern that looks more like my lesion than looks like normal. And then the endometriomas show very few epithelial cells. So this teeny little line there is the epithelial cells and a predominance of fibroblasts.
So we then did some single cell data to highlight the resident and infiltrated immune cells in the endometrium. And we saw that the non-resident infiltrated immune cells displayed different endometriosis specific signatures or different express genes than the endometriosis patients. So this is the control endometrium on the top lines and the endometriosis patients' endometrium on the bottom lines. And you can see there's differences in their peripheral NK cells, their B cells, their monocytes, and their infiltrated macrophages. So there's definitely a difference in control endometrium and endometriosis endometrium, even in patients that are being well suppressed. So these patients were being suppressed either because they were on contraception waiting for their tubal ligation, or because we were treating them with progestin therapy for their endometriosis. We then did spatial transcriptomics where we took our tissue and did hybridization using zenium spatial transcriptomics. And we saw that endometriosis affects the endometrium by affecting not only its cellular composition, but also it's gene expression.
So you can see in our endometriosis patients, there are significantly more stromal cells, I'm sorry, fibroblasts than epithelial cells. And that the differences in the fibroblast subpopulations show an increase in proliferation and differential gene expression between the control, endometriosis control and endometriosis endometrium, sorry, too many endos. Alright, so all of our patients were on progestin therapy. And we did this because we know that there are lots of changes in the menstrual cycle. And actually there was a recent paper or paper that came out in 2020 that looked at single cell transcriptomics along the menstrual cycle, and they found seven different cell types in the human endometrium. Again, this is their single cell data, so all their little piles of Legos. But you can see the transcriptomics changed significantly through the menstrual cycle. And so our hope was when we put them all on the same therapy, that we would have them in the same spot so we could actually compare them better because there is a significant difference in the transcriptome as we go through the cycle.
And you can see there's actually an abrupt change here at the window of implantation, which is important as well. So Dr. Cameron. So Dr. NE's oldest brother just recently put out a paper looking at the real world perspectives of endometriosis. And what I liked about this paper was that it was a review of all the different things going on to look at endometriosis, and it's my epitome of a team sport type of paper. And in this review, he saw key features of three different endometriosis lesions, both the superficial, the deeply infiltrating and the endometriomas that was seen with single cell RN aeq. So he took all the data out there and compared what he saw. And we saw that superficial endometriosis display epithelial stem cells similar to the eutopic endometrium. So maybe these superficial lesions are more similar to the endometrium in those patients that deeply infiltrating endometriosis has features of neo angiogenesis and immuno tolerance to the microenvironment, as well as development and survival of nerve signaling. He looked at superficial peritoneal endometriosis and DIE sharing similar fibroblasts and stromal genes, but differing from the endometriomas and that endometriomas are mostly fibroblasts and pro-inflammatory with t t-cells, NK cells, and macrophages. So similar to some of the data that we had.
So I'm going to take a little bit of a spin off of this for a second because I want to look at, I thought this paper was very interesting because it looked at single cell transcriptomic profiling of the luteal phase right around the window of implantation, and it compared women who are controls to women with recurrent implantation failure. And the reason I thought this was interesting is because they looked at the endometrial dynamics of fertile women across the window of implantation and compared it to endometrial dynamics in women with recurrent implantation failure. And they uncovered very similar to all we've heard today, a hyper inflammatory microenvironment perhaps leading to dysfunctional endometrial epithelial cells in patients with recurrent implantation failure. And they provided a platform for therapy for recurrent implantation failure. But what's really interesting, oops, I always keep pressing the wrong button, is again, here come back our NK cells and our T cells and how they're elevated in patients with recurrent implantation failure, just like what we saw in our patients who have endometriosis. Could this be part of why patients with endometriosis have infertility? Since we don't understand why patients with endometriosis have infertility, especially when their anatomy is theoretically not distorted.
So actually one of our REI fellows did a retrospective study looking at the receptivity assays done in control in endometriosis patients and in program cycles. And this was a endometrial biopsy done supposedly around the time of the window of implantation based on progesterone given during these cycles in these program cycles. And what you can see is that the endometriosis patients, less of them, were receptive at the time that they were supposed to be receptive at the window of implantation, and most of them were pretty receptive. So something is going on that this endometrium is not ready for implantation. Again, could this be part of why we have problems with infertility and endometriosis patients even when they just have just have superficial peritoneal lesions? And I'm sorry to use superficial, Dr. Seskin peritoneal lesions. Excuse me.
I don't want to get in trouble. He used to be my attending. And by the way, it's been 20 years since I've stood on this podium 20 years ago. I graduated from this residency program, and the last time I started in this podium was to do an m and m on an obstetrical hemorrhage. So I'm having a little bit of flashbacks with all of you guys out there, but hopefully I'm doing better than I did my chief year. So finally, I'd like to talk about validating these single cell findings in 3D cellular models. So wouldn't it be great if we could make cellular models? And I know we're going to talk about this more today where we could try to test some of these things that we are talking about today so that we don't have to test them in our patients. And so what we did was try to make some functional tissue and we made some organoids, and then we tried to validate what we saw in our single cell analysis in our organoids.
So we made these organoids and we saw the same things in our organoids that we saw in our tissue that we got from the patients. We found very similar spatial transcriptomic findings to what we were getting from our patients. So hopefully these organoids and some of these models that we are going to be talking about today will be a way for us to test these treatments, not on our patients, but in our labs, so that we can make personalized targeted therapy for our patients. So what has single cell taught us about endometriosis? There are definitely differences between control and endometriosis, endometrium in a cellular composition and end gene expression. Endometriosis patients have novel immune complexes and immuno modularity signatures in eutopic endometrium that differ from control. Patients endometriosis and endometriosis endometrium have immunomodulary, microenvironments endometriomas are very different from, excuse me, peritoneal lesions suggesting they may be a distinct type of endometriosis.
Patients with recurrent implantation failure have delayed windows of implantation and hyper inflammatory microenvironments with similar immune cell types to those expressed in endometriosis patients. These findings can be replicated in 3D organoids and maybe these organoids can be used to test treatments or to personalize treatments for endometriosis. So most importantly, endometriosis is a team sport. It needs to be treated that way. We need all of the PhDs and we need all of the patient advocates, and we need all the moms and all the physicians and all the surgeons to work together so that we can bridge medicine with cutting edge technology and science. And so this is my team. Whoops, sorry. This is my team over at Yukon. This is Elise over at Jax. We're just this teeny little five minute walk except for when it's really cold, but it's also the World Endometriosis Foundation. It's also the state of Connecticut and Jillian Gilcrest who felt that endometriosis was important enough to do state funding for it and to advocate for state funding and for the Endometriosis foundation and other foundations like that that work towards helping our patients. And this is our most recent snowstorm at Yukon. Thank you.
