Journal Club Global: Non-invasive Diagnosis of Endometriosis

And some backstory to this, Martin and I have known each other for a little bit. We actually met on Twitter, engaging on the topic of endometriosis and fertility and sterility had been really kind of pushing this topic of webinars and collaborating with different organizations and journals and Martin and I kind of had a natural connection. So we did something on malaria anomalies probably a few months ago now that was just really well received.

There's tons of attendance. It was kind of how Europeans and North Americans are classifying malaria and anomalies. And we thought this is great.

We should continue doing this. So this is actually the very next event. This is the second one.

I think we've hit the go button. So we are now live a little bit early. So there are a few people on.

And I think if everyone's okay, who's currently on and has joined early, then we'll continue just chatting about how we know each other. And so give a little bit of a backdrop to the panelists and the moderators. And for everyone that's wondering, it's the first time that I've met Valerie and Pietra and I know ourselves.

So sorry, continue Pietra. Well, hi, everyone who's now joining. Our apologies that we're eight minutes early, but you can get see how the sausage gets made, so to speak, and hear kind of the backstory and how these events come together.

So the Malarian Anomalies joint webinar with FNS and ASRM and the BSGE was our first event. And we just had a really positive response. There was a lot of people that were interested in hearing how North Americans and Europeans are doing things differently.

And we thought, you know, there's so much more overlap beyond just surgery, what the BSGE does and FNS does. And endometriosis kind of seemed like the next logical space to explore. And rather than talking about how we do laparoscopy different or how we do oral medication management of endometriosis differently, we really thought kind of the next big frontier is going to be talking about non-invasive diagnosis, because I think we all have this very strong belief that diagnosis is therapy.

And being able to put a name to what men and women are feeling when they experience these symptoms is really, really powerful. So we wanted to really focus this session on that. And I'm so glad that you agreed to do this, because I know you've done so much work on biomarkers, both as a fellow and now as a faculty at Yale.

And Suzanne, I've followed you on Twitter for quite some time now as well. And I know you've done so much education on ultrasound as a way of early diagnosis of endometriosis. And there's so much going on in this space.

So we figured you two would be the perfect ones to educate us on what's coming, what's next, what can we all expect beyond just sticking a laparoscope in someone's belly button for half an hour. So thank you. All of that to say, thank you for joining.

Thank you so much for the invitation. One thing, one backdrop to this story is, I think Suzanne and my paths may have crossed, because I worked in Southampton from 2007 to 2009 intermittently in the women's health department. And I know Suzanne, I think has been in Southampton quite a long time.

I was obviously a very baby, baby, baby doctor back then, and was probably asking permission to talk to Suzanne back then. But it was, yeah, I had a great time in Southampton. And so I'm not sure, I think Suzanne may have logged in, but be finalizing a few bits before the webinar kicks off in five minutes.

But I can highly recommend Southampton as a unit, as a great unit for endometriosis and all other specialties. And then Suzanne, I just made you co-host right now. Lovely.

Thank you so much. Can you see me now? Brilliant. That's good.

That's, that's lovely. Well, thank you for the kind introduction as well, Martin and Pietro. And nice to meet you, Valerie.

And I think this has to be the single most important thing at the moment is the earlier diagnosis of endometriosis, because it's such a massive problem. And we've all, we know so much that isn't widely shared. And this sort of a platform is fantastic to be able to share what we know about how we can bring forward women's diagnosis, because seriously, eight years is ridiculous to be able to diagnose anything.

And so I really look forward to sharing my video with you guys and then taking questions afterwards to see what you make of it. I think that you will agree if you don't already agree that ultrasound has and will have a huge role to play for women with endometriosis or other kinds of pelvic pain. And it's a question of how on earth do we get this knowledge disseminated? How do we train enough people so that everybody when they have pelvic pain has access to a high quality ultrasound? And I look forward to hearing your thoughts about that.

It's a monumental task. But it takes great clinicians and leaders like yourself, Suzanne, in the field of ultrasound to hopefully spearhead the thought process and design the programme of education to enable high quality ultrasound to be readily available to all the patients that we see. Yeah, yeah.

Numbers are tallying up. My inbox was flooded on a Monday with my weekly update from PubMed of endometriosis publications. And I've seen that Valerie, you have a study protocol on, I think, is it pregnant? Is the DMJ open? Do you want to tell us all a little bit about that? Of course.

Oh, thank you. So this is a trial that we are spearheading at Yale, but also working with a couple of other collaborators. There has been some work looking at pre-treatment in women who have endometriosis who are undergoing in vitro fertilization, whether or not pre-treatment with combined oral contraceptives for six weeks, which is a pilot trial versus Lupron for which GnRH agonist for three months prior to undergoing IVF, whether or not that improved IVF outcomes, specifically looking at clinical pregnancy rates.

One of the limitations of both of these studies was really the fact that it only got as far as looking at clinical pregnancy rates. And so we wanted to also take a bit of a step further and look not only at CPR, but also like birth rates and whether or not there were any changes or improvements rather in any potential antepartum outcomes associated with endometriosis. And actually there was a new Cochrane review that came out that really questioned the utility of GnRH analogs or suppressive therapy prior to IVF.

So I'm actually even more excited because I'm really hoping now using a GnRH antagonist, which has a shorter onset and offset. So we're only doing this for two months pre-treatment, whether or not this will really help add to the literature and really show that it really is beneficial and could this now be the new standard of care prior to IVF. So if anyone has any questions or has any patients that may be interested, feel free to email me.

I'm very excited about this. We've got two minutes left and I haven't read the full protocol, but does it include patients with adenomyosis as well? Does it exclude those? It's not an exclusion, but in order to be included, you have to have endometriosis either, of course, a bit of a downside laparoscopically diagnosed or an endometrioma by ultrasound. And so I know both Susan and I will be talking about noninvasive diagnosis, but for the purpose of the study, it is based off of what's currently the gold standard.

And I'm sure we'll both talk a little bit about the limitations of the current gold standard, but gold standard for now would be laparoscopically diagnosed or ultrasound diagnosis with an endometrioma. And for everyone who's joining, you're probably walking into a conversation. No, we have not started yet.

We have one minute to go. We're just bantering around because we hit the start button a little bit too early, but to set the stage, we're going to, Martin's going to welcome all of us in a moment. I will introduce Dr. Flores and then Martin will introduce Dr. Johnson.

And then Dr. Johnson will spend some time sharing a video that she's put together about how she uses ultrasound for the diagnosis of endometriosis. And then Dr. Flores will have some slides for us to share about her work on biomarkers. And at the end, we will have a question and answer session.

So there will be an opportunity for us to ask questions of them. If you have questions, you can certainly include them in the chat, particularly in the Q&A section. If not, Martin and I have some thoughts that we'd like to explore with them.

We're just going to hold tight for another minute or so, and then we'll get started. You have not missed anything. You have not logged on late.

We are early. I think this is, we need to be kind of applauded for starting early and being ready and prompt and ready to go. So yeah, thankfully people that log on on the dot of nine or nine this time, four o'clock Eastern time, will be ready to start.

And I think my clock's at nine. So on behalf of the BSGE, my name is Martin Hirsch. I'm a consultant gynecologist, endometriosis specialist based in Oxford in the United Kingdom.

I am recently appointed senior council member of the BSGE for awards and bursaries. So very pleased to announce that for the first time. And on behalf of the BSG, we're really pleased to be partnering with Fertility and Sterility for yet another webinar.

I think there's only things to be gained and learned from collaborative efforts like this in terms of the spreading of knowledge from different parts of the world that I'm really proud to be part of. And so on behalf of the BSGE, I'd like to say thank you for joining and I'll hand over to Pietro for a few words. Hi everyone.

My name is Pietro Bordoletto. I'm a reproductive endocrinology fellow at Weill Cornell for another eight days before I graduate from fellowship. But in my other roles, I serve as the interactive associate in chief for Fertility and Sterility and the media editor for Fertility and Sterility reports.

And I just want to thank Martin for again welcoming Fertility and Sterility back to host a joint webinar where we team up with our European colleagues to explore topics that are relevant to all of us. Martin is a surgeon. I'm a reproductive endocrinologist and surgeon and endometriosis is at the intersection of all of that.

And tonight we're hoping to spend some time sharing with you a little bit about what is on the horizon in terms of non-invasive diagnosis. I think long gone are the days where we need a laparoscope and an abdomen with general anesthesia to be able to confirm what we think. And really the next step forward for going from diagnosis to early intervention is going to be the work that's being done for non-invasive workup.

So today we have two experts. We have people from both the United States and from the UK that are going to tackle this issue from slightly different angles. Dr. Flores, who I have the pleasure of introducing, is a assistant professor of OBGYN and reproductive sciences at Yale.

And she's also a reproductive sciences development program scholar. Her research, and the reason why she's really here to talk with us today, is she's working on determining the molecular mechanisms that are involved in the path of physiology of endometriosis in order not only to speed up the time to diagnosis, but also develop novel therapies at hopefully better managing this disease beyond what we have today. And Martin, I think you're going to introduce Dr. Johnson, who's going to take us through kind of another approach to non-invasive diagnosis for endo.

So we're really pleased to be joined by Suzanne Johnson, who's an associate specialist down in Southampton and an international leader in the non-invasive diagnosis of endometriosis by ultrasound. She is research active and specializes in the field of endometriosis diagnosis amongst other things, including cesarean scar pregnancy and risk prediction for adnexal masses. She is the founder and author of a website called gynecologyultrasound.com that aims to spread awareness and improve education around the use of ultrasound.

And without much further ado, I'd like to share her presentation. And the reason why it's being shared as a prerecorded session is that the videos come across without any glitches but Dr. Johnson has joined us live. And perhaps, Suzanne, you'd like to introduce your presentation.

Lovely, Martin. Thank you very much. Thank you for inviting me to talk to you about ultrasound and the diagnosis of endometriosis.

I've made a video and we're sharing it as a video because the ultrasound clips of video don't pixelate if you do it that way. So I'm sorry, it is a prerecord, but I will be right here. I'm very happy to answer any questions that you may have at the end.

So take it away, Martin. Thank you. Here we go.

Let's share screen. Martin, I just made you co-host. I don't know if you were on there before, so I think now you should be able to share.

Fantastic. Take two. Technology is clever, but it's sometimes not quite clever enough yet, is it, when we're doing these live Zoom meetings and there's often a tiny little hiccup.

But soon enough, this video will start. And then I apologize if for some of you it's repetition and things you already know, but I'm hoping that everybody will find something useful in there. And bear with the beginning.

It's probably a little bit simplistic, but you'll suddenly see why I've started simple. And getting the patient comfortable is an incredibly important part of that. If the patient's in pain, you're not going to see very much.

So how to get the patient to experience the examination as relatively comfortable is incredibly important. So there's a little bit about that in the presentation as well. You want me to try to share a screen? Would you be able to? Yes.

Apologies. I just need to. I don't know.

This is the first time I've done this, so it might work. It might not. We can see your screen share and we see the PowerPoint presentation.

Okay. And sorry, I haven't done this before. How do you press play? That's the question.

Maybe I have to press play on the file on the desktop. Do I? If you click to do presenter. Yeah.

Presenter view. I think from there it should automatically play. Let me see.

Use presenter view. Presenter view or slideshow. If you go on the PowerPoint and click view and then do slideshow, from there it should automatically let you play the video.

Sorry about this, guys. I've never done this before. If we need a little bit more time to sort out the play issue while we could potentially have Dr. Flores.

Why don't we do that? I think just in the interest of sorting out the technical issue, if Dr. Flores, if you're okay with going first, would that be all right? No problem. Perfect. All right.

So Dr. Flores, maybe you can introduce your talk and then we'll listen to have you present first. Perfect. All righty.

Just working on sharing my screen. Can everybody see my screen? We can see your slides perfectly. Perfect.

Okay. So thank you again, Pietro, for that introduction. I'm Valerie Flores, Medicine Assistant Professor at Yale University School of Medicine.

And this is near and dear to my heart. Actually, one of my aunts had endometriosis and how she presented was with infertility. Around the time that she told me about her story, I actually met Hugh Taylor, who's my research mentor.

So I've been very invested in not only minimizing the time to diagnosis, but also maximizing how we choose therapy for a given patient. So with that, I'll start. I'm going to be talking about noninvasive diagnostic approaches for endometriosis and also including a bit about the diagnostic delay and how viewing endometriosis as a systemic disease can also hopefully from a clinical perspective, as we're developing better noninvasive diagnostic approaches will ultimately help in minimizing that time to diagnosis.

My one disclosure is that I do receive speaking honorarium from AbbVie. However, I will not be discussing any of their drugs specifically. As many of us know, endometriosis is defined as endometrial-like tissue outside the uterus.

And the most commonly accepted theory is that it's due to retrograde menstruation. However, I'll talk a little bit about some other theories and how it really helps us understand the disease on a much more systemic level. Patients can present in various different ways.

Some present with pain, some present with infertility, and some can actually be asymptomatic. And in studies, really looking at stage of endometriosis and how that correlates with symptoms, it actually doesn't correlate well. And so this is really highlighting the fact that it really is a heterogeneous disease.

And so we really need better approaches to how we diagnose and think about how some of the symptoms impact presentation. In terms of epidemiology, it affects up to 1 in 10 reproductive-aged women, up to 80% of women with pelvic pain, up to 50% of those with infertility. And while it has traditionally been characterized by its pelvic manifestations, it's important to recognize that it's profoundly more than a pelvic disease.

It really is a chronic systemic disease with multifactorial effects throughout the body. And the healthcare costs in the U.S. alone exceed $22 billion. It also really affects women at a formative time, critical to their education and early careers.

And the true cost really includes the hindered potential of affected women. The diagnostic delay can be up to 11 years from symptom onset to diagnosis and treatment. And why does that happen? So sometimes the symptoms can be very nonspecific.

They can be associated with other disorders. Pain in and of itself can be considered subjective. And social norms many times can inhibit the conversation.

Patients either feel uncomfortable describing their menstrual symptoms with their providers, or if they have a very strong family history of endo and may not know that it's endometriosis, sometimes their pain is even normalized from a family history perspective. And it goes without saying that it's very unfortunate to see that over 65% of women are initially misdiagnosed and almost 50% see over five providers before receiving the correct diagnosis. And so again, the question I ask is why are the symptoms nonspecific and why is endometriosis difficult to diagnose? And the answer really requires us to shift our perspective on how we see endometriosis and really looking at endometriosis as a complex systemic disorder.

One of the examples of the systemic nature of endometriosis really comes from work assessing the role of stem cells in the pathogenesis. So as I mentioned before, the most commonly accepted theory is that of Sampson's, which is retrograde menstruation. If a laparoscopy were to be done on all women at the time of their menstrual cycle, up to 90% will have retrograde menstruation, but only 10% actually have the diagnosis of endometriosis.

So something else must be happening. Not only that, we can see endometriosis in women who don't have a uterus and actually can even be seen in men who have been exposed to high levels of estrogen. So this led to really looking at the role, the potential role of stem cells.

Bone marrow derived stem cells in particular have been found to be able to, the mesenchymal component has been found to really be able to turn into an endometrial derived stem cell, and that can either get lost and ultimately end up in the pelvis and turn into an endometriotic implant, or in women who have the disease established already, what can happen is these stem cells, although they may need to go home to the uterus, instead actually go to the endometriotic implant and further promote disease progression. Endometriotic lesions themselves can also produce stem cells, and these lesions can actually end up tracking back into the uterus. However, this occurs incorrectly and may be one mechanism by which endometriosis is associated with infertility.

There has also been work looking at small molecules, and I'll talk about them a little bit more later in the presentation, but looking at things like micro RNAs and the role of inflammatory cytokines and how they can really have this, these long range effects that go beyond the lesions in the pelvis, they can have effects in the liver, brain, lung, adipose tissue, and also within the endometriotic lesions themselves and the endometrium. Some specific examples of the systemic effects are, it's commonly been seen in women with endometriosis that they have a lower BMI. The misconception was really that being thin puts you at risk of having endometriosis, but actually it's the opposite.

The endometriosis in and of itself puts women at risk of being thin, and we and other groups have shown that there's induction of hepatic or anorexogenic gene expression, and there's micro RNA-mediated change in adipocytes as well. Mood disorders. Many times we see patients who have anxiety, depression, also have a diagnosis of endometriosis.

A lot of times these patients are unfortunately being considered as needy patients or having poor pain tolerance, but it's truly the disease and how it manifests in the nerve processing. And then of course, the inflammation, both estradiol-mediated and also due to aberrant micro RNA expression. There's also altered progesterone responsiveness and altered immune cell function.

So what we see in the pelvis is really part of the disease. It really is the tip of the iceberg. And in knowing these systemic effects that endometriosis can cause, instead of just thinking about this disease as causing pelvic pain or infertility, really we need to look at how it can contribute to low BMI, how it can lead to anxiety, bladder dysfunction, cardiovascular disease.

So really looking at these long range effects and how it affects other organs beyond the pelvis. And why is this important? Because again, there's such a large diagnostic delay that if we can shorten that delay from a symptom perspective and how we think about the disease process, and also as I'm going to get into shortly from a non-invasive approach to this disorder, that really will reduce the uncertainty, the discomfort, and the later complications associated with endometriosis. What do we have available then in terms of non-invasive biomarkers? Because currently the gold standard has been surgical diagnosis, but that in and of itself contributes to delays.

So really focusing on how can we non-invasively approach this disorder. I know Dr. Johnson will be talking about imaging. I was going to be focusing on potential endometrial biomarkers and also serum biomarkers.

With respect to endometrial biomarkers, there are several. There was a Cochrane review actually in 2016 and Estrey also updated its guidelines in 2022. There have been assessments of angiogenesis and growth factors, cell adhesion molecules, inflammatory markers, myogenic markers, nerve growth markers, and also tumor markers like CA125, but they're not recommended.

And the reason why is just there's a lot of heterogeneity and there's variable sensitivity and specificity, even when combining some of these biomarkers. One newer one from Dr. Lessie's group actually is looking at BCL6 or B-C-L-C-C-L lymphoma 6. It's associated with cellular proliferation. And up until recently, it was not known what potential role it could have at the level of the endometrium.

So Dr. Lessie's group looked at a couple of different things. First, it was just to assess whether or not BCL6 was present in the endometrium. And then also looking at whether or not it was specifically noted in the proliferative phase or the secretory phase.

And then of course, looking at if there was a difference between women with and without endometriosis. So on the left-hand side, we're looking at the fertile control. So these are women who had no history of endometriosis, no history of infertility.

And this was then compared to women who had undergone laparoscopy for presumed endometriosis and then ultimately diagnosed with endometriosis. And it was found there was significantly higher expression of BCL6 specifically in the secretory phase in women with endometriosis compared to controls. To really test out whether or not BCL6 could be used as a marker of endometriosis, there was a second group of individuals, again, fertility controls.

These were women who were undergoing surgery for tubal ligation, also underwent an endometrial biopsy at that time compared to women actually with unexplained infertility who ultimately underwent a laparoscopy. And you can see that with the fertility controls and the unexplained infertility, oh, my marker is not showing up, sorry. So for the fertility controls and the laparoscopy controls who were unexplained infertility patients who underwent laparoscopy as part of their workup, there was still lower expression of BCL6 compared to the women who were ultimately diagnosed with endometriosis.

And this was using an age score. So taking into consideration both the intensity of staining and the percentage at a given intensity. So this actually really represents a novel endometrial marker for diagnosing endometriosis.

It was associated with a 96% sensitivity and a 93% specificity. The one point of caution is, again, these were very clean samples. And so this is also taking into account the fact that women who had, for example, polycystic ovary syndrome were not included.

And so we do need more prospective data, but this is actually really one of the first endometrial biomarkers that may be able to predict women who have endometriosis. And there's ongoing work looking at how it may predict response in women who are undergoing IVF. In looking at serum biomarkers, the three primary ones have been looking at angiogenes markers like VEGF, pro-inflammatory cytokines, and also looking at CA125.

Again, while we frequently do see that these factors are elevated in women with endometriosis, even when these markers have been combined, they haven't really been sufficient for being able to either reliably diagnose endometriosis. And again, it's due to variations in the specificity and sensitivity of these tests, but not to fear. So they're also, that's why I'm here to tell you a little bit more about some new serum biomarkers for diagnosing endometriosis and specifically looking at the role of microRNA.

So I know I introduced that topic a little bit earlier. What we have been seeing is that it does, these microRNAs are having an effect on the disease process, but the question now is why even think about looking at microRNAs? So these microRNAs are non-coding RNA molecules, and in various conditions, they have been found to be differentially expressed. They are short 22 nucleotide sequences that really can either cause repression or degradation of messenger RNA.

And they're quite stable, both in the tissue and in the serum, they're actually quite stable molecules. And increasing work, both in endometriosis and in the cancer world, has really shown that they have been differentially expressed. And while there have been several groups looking at it in endometriosis, I was going to focus a little bit on some of the work that we have done in the lab.

So again, microRNAs have been found to be differentially expressed in endometriosis and other conditions. What we wanted to look at is really how many and which ones were the most significantly differentially expressed. So on the right hand side, these were some microRNAs that were actually increased in expression.

So this was a microarray that analyzed over 30,000 microRNAs. And then we specifically looked at those that had over a tenfold difference. The majority have been found to be upregulated in endometriosis.

And then the two that we looked at that were downregulated were microRNA 6755 and microRNA 3613. So this is obviously interesting to see. And as I've noted before, has given us a lot of information with respect to how microRNAs are involved in the disease process.

But the question is, can they be used for diagnosis? So can we really say that, yes, they're upregulated and it is different from women who don't have endometriosis and can we develop that into a test? So in looking at these various microRNAs, how we were deciding whether or not it could be used as a test was looking at the area under the curve. So this was from an ROC curve analysis. There were, as I mentioned, there were several microRNAs that were differentially expressed.

However, it was only upon combining three in particular. So microRNA 125, 451, and 342. Now we found that the area under the curve was one.

So this is saying that with essentially 100% certainty using this combination of three microRNAs, we can accurately diagnose women with endometriosis. The control group were women who were undergoing surgery for different indications, laparoscopy, who were ultimately found to not have endometriosis. This study in particular was done in women who had stage three and four disease.

And so this is obviously not representative of all stages of endometriosis. And so we decided to perform now a second study. This is actually a prospective study, including 100 women, 50 controls, 50 endometriosis cases.

The controls again, were women who were undergoing laparoscopy for other indications. And now here we were able to find actually now six microRNAs that were differentially expressed in endometriosis versus controls. And also now looking on the right-hand side, looking at whether or not these microRNAs were differentially expressed based off of menstrual cycle phase, which they were not.

But again, in looking at these six microRNAs, we were able to see that they were differentially expressed between endometriosis and controls, but wanted to see again, either one, was there one particular microRNA that could be used to accurately diagnose endometriosis, or was another combination approach more beneficial. And so here again, I'm showing the area under the curve. It was now looking at these six microRNAs that once again validated our prior work where the train is actually in this original trial, the 50 women with and without endometriosis.

But then we'd actually took the 48 subjects from the prior trial and did a test to see whether or not, again, this test was truly accurate. And now the area under the curve was still not 100% now, but was 0.93. So this is really exciting because this is now one of the first available serum tests that can reliably diagnose women with endometriosis. Again, this sample size, although 100 was still in the grand scheme of things, still a small study.

And so there's ongoing trials now looking at validating this even further in women with undergoing surgery for endometriosis. I next wanted to switch gears a little bit to talk about biomarkers for predicting response to medical therapy. Pietro, sorry, do you want me to take questions now or wait till the end? Because I did see a couple of hands go up, so I wasn't sure.

We'll do questions at the end. This is so fascinating. I'd like you to continue the chat.

Thank you. So one of the other aspects of endometriosis that I've been very interested in is really predicting response to medical therapy. First line medical therapy has been progestin-based therapy.

However, up to a third of women fail first line therapy. And so this is what we term progesterone resistance. But I think what happens sometimes is we immediately shift over to second line agents.

But the question that I have had, and I'm sure many others have had, is why are women failing progestin-based therapy? What is the actual cause? Is it decreased progesterone receptor? Are there micro RNAs mediating progesterone resistance or methylation of PGR, for example, that then leads to decreased receptor expression, or could other factors be involved? So I clearly can't take credit for that initial hypothesis. I was actually looking at some work from Judish, and she had actually looked at an endometrial microarray and had found that it was specifically in the secretory phase of women with endometriosis. Women with and without endometriosis where there is the most differentially expressed genes in the endometrium.

Not only were these genes associated with mitosis and proliferation, which is normally what progesterone is blocking when we think about estrogen mediated proliferation and growth, but also specifically looking at progesterone target genes. And these were also found to be decreased in women with endometriosis. This is important from the perspective of treatment where we want to cause lesion regression or decidualization, but also important at the level of the endometrium when we're thinking about what's necessary for implantation, which is also appropriate endometrial decidualization.

So from this work, what I wanted to look at was, is this decreased target gene expression of progesterone due to alterations in the progesterone receptor? And could we ultimately use this to help predict response to progestin-based therapy? So to answer this question, I completed a retrospective cohort study in women who were undergoing surgery for endometriosis. I had 52 women who were included. In order to be included, one had to undergo a laparoscopy and have tissue available for PR expression levels, and also whether or not I had information on if they responded to progesterone-based therapy or not.

I also used the age score for quantifying expression and defined response as not requiring surgery or not requiring any change in therapy. And on the right-hand side, what you can see is that in non-responders to progesterone-based therapy, there was significantly lower levels of progesterone receptor compared to those who did respond to progestin-based therapy. So this is interesting because a lot of the work looking at endometriosis and PR is really focused on the fact that PR has just been found to be lower in women with endometriosis compared to women without endometriosis.

But no one had really looked at whether or not there was differential expression of PR even within endometriosis patients, specifically looking at those who did or did not respond to progestin-based therapy. But what I next wanted to see was whether or not we could use PR status then to predict response to progesterone-based therapy. So this was, again, taking advantage of an ROC curve where I was able to actually develop two different cutoffs, so two different thresholds for predicting response or lack of response to progesterone-based therapy.

To be considered a responder having high PR expression, that was defined as an age score of over 80, given its 100% specificity and positive predictive value. And so in women who had a high age score, who their lesions had a high age score, 100% of them responded to progesterone-based therapy. Admittedly, it's a low number, it's seven women, but I think that, again, highlights the fact that in endometriosis, we see that there is a lot of progesterone, or that there is progesterone resistance, and up to a third of women do fail progesterone-based therapy.

So really, I was focusing then on this low PR group, and so this was defined as an age score of less than or equal to five. Again, given its reassuring sensitivity and negative predictive value in here, what we're seeing is that in women's lesions who had low PR expression, the chance of responding to progesterone-based therapy was almost zero, it was 6%. So 94% of women who had low PR expression in their lesions would have not responded to progesterone-based therapy.

I wanted to then show this in vitro, and so looking again now at the endometrium of women with endometriosis comparing to a cell line. This is a normal cell line of just human endometrial stromal cells, and what I did was I provided exogenous estradiol health constantly at 10 to the negative seven, and then increased concentrations of progesterone. And what you're seeing is that, as would be expected, progesterone is able to block estrogen-mediated proliferation, and you see that proliferation essentially drops to zero with increasing levels of progesterone, that's a normal response.

And then now we're looking at a progesterone-responsive subject where, again, you see that with increasing levels of progesterone, it's able to carry out its usual function of blocking estrogen-mediated proliferation. Now this is in contrast to a progesterone-resistant subject, so same cell line on the left just for reference purposes, but what you're seeing is that with the progesterone-resistant subject, even with increasing levels of progesterone, you never really drop down to zero proliferation, there's still some baseline proliferation happening. So the last study that I showed you was looking at surgery, which obviously is invasive, and this is now looking at the endometrium, which would require only an endometrial biopsy to predict response or lack of response to first-line therapy.

I next wanted to look at this now in a murine avatar just because it's a little bit harder to randomize patients to receive progesterone-based therapy or not, so I took advantage of the murine avatar to really look at, again, progesterone responsiveness. I utilized eight-week-old non-SKID mice, so immunocompromised mice, and this is just an example of human endometriotic lesions that were implanted into these mice. Before implantation, and the reason why I used endometrioma is just because they're much larger portions of tissue.

I also looked at the PR expression and defined the lesions as having either high or low PR expression. I did not over-reactomize these mice, but after two weeks post-transplantation, treated them with either progesterone-based therapy, medroxyprogesterone acetate or vehicle, and after one month of treatment, ultimately sacrificed the mice, measured the lesions, and collected them. This is just a representative image of a lesion that had low PR expression compared to a lesion that had high PR expression, and what you're seeing is that the low PR lesion is larger compared to the lesion with high PR expression.

Then, in specifically looking at treatment response, the lesions that had low PR expression really did not shrink in response to progesterone-based therapy, which is in contrast to now lesions with high PR expression, where the lesion is almost completely resorbed. So, just further highlighting this progesterone resistance and how we are able to predict it by looking at progesterone receptor expression levels. In terms of what that means for patients, when progesterone-based therapy is failing or when a patient you know has had surgery before or at least has an endometrial biopsy available, if you see that either one in vitro there's lack of decreased proliferation or you see that PR is low, moving on rather quickly to second-line agents like GnRH analogs, androgen derivatives, and aromatase inhibitors.

And so, to summarize in terms of clinical implications for patient care, so one is really looking at the fact that there's a varied presentation when it comes to endometriosis. No two endometriosis patients are alike, but in really looking at the systemic manifestations of the disease that will hopefully help facilitate diagnosis and shorten the time to diagnosis. Looking at microRNAs as a novel non-invasive approach to diagnosing endometriosis, again with the goal of minimizing existing diagnostic delays.

When it comes to treating endometriosis, utilizing progesterone receptor status for predicting response or lack of response to first-line therapy, and then really focusing on precision medicine to again help minimize just a lot of the negative consequences of the unrecognized disease and untreated disease. And with that, I'm happy to take any questions. You know, after a talk like that, you just become so much more thankful for physician scientists who can actually connect the dots from what you were doing in the lab to what's happening at the bedside.

Thank you for that. That was outstanding. I'm going to hold off on questions for now.

I'm hoping Dr. Johnson was able to sort out the video issue from earlier. I mean, I certainly learned a lot. So thank you.

Okay, there we go. So we're going to have Dr. Johnson play her video, which includes ultrasound clips. And at the conclusion, we'll take some questions.

Lovely. Let's see if it works. Hi, this is a video on the non-invasive diagnostic approach for endometriosis ultrasound, a webinar for the BSGE in fertility and sterility.

My name is Suzanne Johnson, and I'm a gynecologist from Southampton. There are long delays in the diagnosis of endometriosis and worldwide, it's currently around eight years. Many women consult their GPs many times before they're referred.

And it's not quite clear what causes the delays. There is significant overlap with other conditions like irritable bowel syndrome. And there are some issues around the normalization of pain in patients and by their healthcare providers.

But also, there's a myth that you cannot see endometriosis on ultrasound. This is just not true, and I hope to show you how. These are some examples of deep endometriosis that was easily seen on ultrasound.

So a routine pelvic ultrasound mainly looks at the uterus and you'd look for adenomyosis, the ovaries, ovarian endometriomas. But you also need to look for deep endometriosis, need to look for mobility, to look for a frozen pelvis, and for site-specific tenderness. Superficial endometriosis will be covered separately.

The National Institute for Health and Clinical Excellence published this guideline in 2017 on the diagnosis and management of endometriosis. And you'll see that in the very early stages, they would just say consider an ultrasound scan. And after some initial treatment and management, the patient might be referred to a gynecologist with a gold standard for diagnosing endometriosis as laparoscopy.

So a laparoscopy is where we put a camera through the umbilicus looking toward... Dr. Johnson, I think you may have muted by mistake and we are losing your audio. This is what their pelvis might look like. Back of the uterus, the ovaries, the tubes, bowel, everything is adhering to each other.

It's quite hard to see exactly what's what. This is called a frozen pelvis. The International Deep Endometriosis Analysis Group published this paper in 2016, and it gave us both a language for how to describe endometriosis, as well as a system for how to perform such a scan.

And these four steps can be done in any order. Language really matters. And the idea terminology was very clear that if you draw a line through the internal os and the torus and a line below the cervix, then the area between the vagina and the bowel is called the rectovaginal septum.

The bit of vagina above the red line, where it attaches to the cervix, is the posterior vaginal fornix. And then there's the pouch of Douglas between the uterus and the bowel. The bowel below the torus, below the green line, is the lower rectum.

The bowel above the green line, above the torus, is the upper rectum. And then bowel above the level of the fundus of the uterus, it would be called sigmoid. And the posterior compartment is affected by deep endometriosis, and common locations are in the posterior vaginal fornix, at the torus, in the lower rectum, in the upper rectum, and it can all become adherent together.

And this is the frozen pelvis. So in the posterior compartment, there's the vagina, the ligaments, and the bowel. But the anterior compartment is another common place for endometriosis, in the bladder and in or around the ureter.

So before you start your scan, you need to have enough time to do the scan. 30 minutes for a standard scan is a good amount of time. 45 minutes for a detailed pelvic scan, as I'm going to describe to you.

You would start the scan transvaginally, unless you're specifically looking for fibroids, or the patient is potentially virgo intact. And you start the scan with the patient having an empty bladder and no bowel prep. You need to make the patient feel comfortable, lock the door, cover them over, be nice, and then use a bed, not a wedge.

Have the patient's bottom at the edge of the bed, and the bed slightly high, and then feet on the stirrups or on a chair. And if the patient has got stirrups, you can sit between the legs, and this is a good position to look for deep endometriosis. You need to be super gentle with patients with endometriosis, because they've already had a lot of pelvic pain, and explain what you're going to do and get consent.

And I warn them in advance that when I'm quiet, it just means I'm concentrating, and I say, I'm so sorry, but I can't, or will I scan? And then I ask them to put their arms by their sides, let the bottom sink into the bed, and let your knees go floppy. This makes a huge difference. And gently and slowly insert the probe with plenty of lubricant on the outside of the probe, angle the probe backwards toward the sacrum, and do a normal systematic pelvic ultrasound.

In patients with endometriosis, I would also do a renal scan. That's because around 3% of them have silent hydronephrosis. The probe orientation, just to show this is my orientation, so that the uterus is here, the bladder is there, and the bowel is behind there.

Some colleagues scan the other way around. So the idea of ultrasound, this is how I do it. I start off with a systematic routine pelvic ultrasound, looking for features of endometriosis.

Uterine position is something we just need to quickly talk about. This uterus is antiverted and antiflexed. This one is retroverted and retroflexed.

This uterus is antiverted, but it's retroflexed at the internal os, and then this uterus is axial. So what can we look for in this scan for endometriosis? We're going to look specifically for an antiverted but retroflexed uterus. And this is the position it's in because it's being pulled backwards by adhesions.

This is also called the question mark sign, and that was just published by Nadine DiDonato, where she found it was associated with adenomyosis. The Muser Group published this paper in 2019 on the sonographic classification for adenomyosis, and you can see some features like a globular uterus, asymmetrical thickening, some small myometrial cysts, some hyper-echoic islands, fan-shaped shadowing, and sometimes some very early budding, you can see there. The Doppler shows a trans-lesional vascularity as compared to fibroids, where it tends to be peripheral.

And you might see an irregular junctional zone or an interrupted junctional zone, and these are more subtle. So this is what it might look like on ultrasound. Here you can see a bulky uterus, antiverted and antiflexed uterus, bulky uterus with a very indistinct endometrial junction.

It's hard to see it at all. The endometrium is about there. And you can see these little small myometrial cysts.

You can see a lot of stripy shadowing. This is a diffuse adenomyosis. Some other times you can see islands of endometrium in the myometrium.

You can see this clearly here. This is severe adenomyosis. And you can put your 3D on and then you would see an irregular endometrial junction with all this budding that you can see, an irregular junctional zone.

Next, we'd look at the ovaries for ovarian endometriomas. And the IOTA group have described an endometrium as a unilocular cyst with ground glass, hecatonicity contents. It's usually unilocular.

It can be up to about four locules. And you'll see that this ovary sits very low in the pelvis. If you then go transversely, you can see that in fact, this endometrium is adherent to the back of the uterus quite low.

And if you compare that to the mobility of the other ovary, this is freely mobile, as you can see there. So you can see there's an endometrioma and there are uterine adhesions. The other thing you need to look at for endometriomas is whether the ovaries are kissing.

And this is in the transverse plane, of course, a bit of uterus at the front. This is a ground glass hecatonicity. This one is anechoic.

You can get anechoic endometriomas. And you can see that these ovaries are adhering to each other. They're also adhering to the back of the uterus.

It's important that if you see endometriomas, that you look for deep endometriosis. And this we learned from Katrin Eksekustos. And many people, even if they don't know how to look for endometriosis, will notice a low ovary.

So when you see a low ovary, you can see it's at the level of the torus. Rotate your probe transverse and gently press. And you can see this ovary is at least sitting on the utero-sacral ligament, if not actually adhering to it.

You'd have to see a video clip. Another thing we look for, of course, is a hydrosalpinx in between the uterus and the ovary usually, or maybe near the ovary elsewhere. This is in the longitudinal and the transverse plane.

And this is a little video clip of that hydrosalpinx. It's probably more of a hematosalpinx. You can see that the contents are low-level echinocytes, maybe ground glass.

And you can see that there it's attached to the ovary with a small endometrioma. So this again is the unilocular hydrosalpinx. It's very big.

It's adherent to the left ovary with a little endometrioma. And the other side's a big endometrioma. And you can see that it's attached to the ovaries before you can even see the uterus.

So you know that this is all very low in the pelvis. There's the uterus. So having done the systematic routine scan, I then look for the sliding sign.

So the sliding sign, this was published by various authors in 2013. And it's a really good addition to a routine scan. This is an antiverted uterus.

My probe is in the anterior fornix. And you can see how beautifully the uterus moves. The uterus moves one way.

And all of the bowel behind the uterus moves the other way. So the bowel is freely mobile on the back of the uterus. And this is a normal sliding sign.

And I'm doing this by just, I've got my probe in the anterior fornix. I'm very gently pressing in and out a tiny bit with the probe. And only rarely do I need to use my other hand on the abdomen to ballot between.

Usually just a little bit of gentle movement there is enough. So then this is an abnormal sliding sign. Here you can see again, I'm pressing gently with the probe that things move, but it all moves together.

The uterus with adenomyosis, the bowel and endometrioma, everything moves together. This is an abnormal sliding sign. So in a retroverted uterus, where the uterus points toward the patient's feet, and you can see here a little bit of free fluid, which is super helpful that the bowel moves freely on the back of the back of the uterus.

That's where the torus is just at the top there. And you can see this is free movement. So a normal pouch of Douglas.

And just to compare with an abnormal sliding sign, this is an abnormal sliding sign. You can see that the bowel is adherent to the back of the cervix, to the back of the uterus, and there's no movement there at all. So we should really add the sliding sign to every routine pelvic ultrasound.

It's quite quick to learn. You just have to concentrate and repeat it. And if once you know how to do this, you'll make a huge difference because this raises a real suspicion of the presence for endometriosis.

Next though, we look for nodules of deep endometriosis. And deep endometriosis is when the lesion extends for more than five millimetres underneath the peritoneum. You then get cyclical bleeding, which results in inflammation and fibrosis.

You end up with these little dark hypoechoic nodules that are surrounded by bright white fibrosis. That's what we're looking for. We can find those in the posterior and the anterior compartment.

And so if we're looking in the posterior compartment first, look behind the uterus. And on this little diagram of mine, you can see here that the probe is in the anterior fornix. This is the view you get looking through the cervix.

But if you put your probe in the posterior fornix, you look behind the uterus, you can't see the cervix, but you can really see the posterior compartment very well. That's what we need to do. So a vaginal fornix, what are we talking about? This is a normal posterior vaginal fornix.

There's a little bit of air in there. That is the level of the torus through the internal os. And I'm going to show you that my probe is in the anterior fornix.

I'm going to show you the anterior lip of the cervix, posterior, and this is the vagina. This is the point where the posterior vaginal fornix attaches to the cervix. So I'm in the longitudinal plane, just going from side to side, just showing you what the normal posterior vaginal fornix looks like.

Just like that. So how do we get from the anterior to the posterior fornix? We need to withdraw the probe a little bit from the anterior fornix, angle it slightly further backwards while encouraging the patient to let her knees go floppy. And then it's quite straightforward.

And if you're gentle, this doesn't hurt at all. So we're just in the longitudinal plane. I'm in the anterior fornix.

I'm going to withdraw the probe a little bit, angle my probe backwards. And here I'm very gently inserting the probe into the posterior fornix. And so the vagina is now overlying my probe, and there's a little bit of free fluid, which is always super helpful.

If you have a good look around, there's some peritoneum and some ligaments, a nice reflection of the pouch of Douglas just there. And here we are in the anterior fornix again. And you can see how when we were in the anterior fornix, we could see the cervix, but when we were in the posterior fornix, we couldn't see the cervix.

So what does forniceal deep endometriosis looks like when the vagina itself is affected by deep endometriosis? This is what it looks like. You could see there's a bit of normal vagina, normal, normal. And then this bit of vagina is affected by deep endometriosis.

You can see it there, little areas of hemorrhage within. This is what it looks like on the speculum examination. You can see that there's some, the little bluish tinge of blood vessels in this slightly irregular nodule there.

So you might look for DE in the vagina itself, but also in the ligaments. And this is a very common place for deep endometriosis. So what are we talking about? The torus and the uterus sacral ligaments.

This again is the view at laparoscopy where this side of the patient is her right, because we're looking toward her legs. It's the other way around to an ultrasound. Posterior aspect of the uterus.

This is the torus and the torus is where the uterus sacral ligaments insert on the back of the cervix. So this is torus, right uterus sacral, left uterus sacral ligament. And these tissues become affected by deep endometriosis very commonly.

This is the pouch of Douglas just here. There's this space between the back of the cervix and the vagina and the bowel, which is just out of view there. So deep endometriosis.

So first you need to see, get good at visualizing the torus. So we're going to be in the longitudinal view where the bladder tissues attach to the uterus, internal or torus. I'm going to go side to side a little bit in the longitudinal plane.

Then I'm going to rotate on this point and that will show me the torus and that's where those uterus sacral ligaments attach. So I'm looking at the torus and I'm now going to slightly go side to side, make sure I'm in the right place. I'm going to rotate round.

This is now the torus. The torus is here at the back. This is one uterus sacral ligament and this is the other.

They're very hard to see when they're normal because it's just a very fine white line like that. Those are the uterus sacral ligaments seen through the anterior fornix. This is then what normal would look like.

So this is the torus here. This is the torus and transverse with normal uterus sacral ligaments. But it's very clear when they're not normal.

Here you've got dense retro cervical fibrosis and this dark area within, this is deep endometriosis either in the torus and or the uterus sacral ligaments. And in the transverse plane, it looks like this. You can see this very dense, thick fibrotic ligaments.

So these are the torus and the uterus sacral ligaments seen through the anterior fornix. This will be quite striking when you first see it. You can see the uterus sacral ligaments through the posterior fornix.

So here the probe is in the posterior fornix. The cervix is up there somewhere. And I'll show you in a minute, this dark line is the vagina.

This white line is the peritoneum. And in a moment, the uterus sacral ligament will appear at this angle. And as I advance my probe onto it, the uterus sacral ligament becomes applied to this.

So the white line is then peritoneum and uterus sacral ligament. I learned this from one of Sophie Piessen's YouTube videos. Very good indeed.

So this is me now doing the scan myself. There's the uterus sacral ligament becoming applied to the peritoneum. And there it goes again.

You can't always see the ligament as nicely as that. It helps when there's a little bit of fluid. So that's the vagina.

That's the peritoneum. And that's the uterus sacral ligament. And so when you have pushed your probe in slightly deeper, very gently, of course, these will be at least in part the uterus sacral ligaments.

So what does deep endometriosis look like in a uterus sacral ligament through the posterior fornix? You can see here again, you can see this is the vagina. This you can see is white fibrosis. And within that, you can see an irregular nodule of deep endometriosis.

To see this closely to the probe, you need to use high frequency, use the highest frequency of harmonics. And then you need to move your focus up so that you're focusing all the beam just here and it will pop out for you. So we've looked at vagina.

We've looked at ligaments. Next, we're going to look at deep endometriosis in the bowel. So this is what normal bowel looks like.

Again, my probe is in the posterior fornix. The uterus has been pushed over to one side a little bit. And there I've got the vagina and the peritoneum, maybe a uterus sacral ligament.

Here is some free fluid. And then you can beautifully see the bowel layers. The outer one is the serosa.

Then it's the muscularis layer, which consists of two fine dark layers separated by some fibrous tissue. Then comes the submucosa and mucosa on one side, submucosa and mucosa on the other side, then muscularis on the distal side and serosa. So that's one bowel wall.

That's the other side. And there's the lumen in between. If you get endometriosis, this is always affecting the muscularis layer here.

So you can see this is what it would look like. Normal muscularis coming in, normal muscularis going out. And you've got this lump of deep endometriosis in the muscularis layer just there.

So when you start your TB probe, you can track this layer. If you pull out your probe slightly away from the cervix and then look for this line just there. And this line, as I said, you've got white serosa.

Then you've got two dark layers, the outer longitudinal fibers, a little bit of fibrous tissue, the inner circular muscle fibers. This is the muscularis layer that becomes very abnormal when it's affected by deep endometriosis. So keep your eye on that muscularis layer.

You have to move your probe slightly toward the left, up and down, left, right a little bit to keep tracking it. And you can see that you can track that muscularis layer all the way around. And you can usually track it to just about the level of the uterine fundus.

And this is what a bowel module looks like. So that's the abnormal muscularis. This is normal muscularis.

And then we've got the submucosa mucosa, the other side muscularis. And you can see the discrepancy in the thickness of the wall. One's very thick, the anterior wall and the posterior wall is always normal.

And you put some color on just to check. If there is a lot of muscularity in this, you need to think of a different diagnosis other than just endometriosis. And it's always this side of the bowel.

It's not that side. So this is a different patient again, probing the posterior fornix. This is the bowel module that you can clearly see with normal muscularis going in and out of it.

But what you can also see is this retrocervical fibrosis. And you can see a nodule of deep endometriosis in the ligaments. You can also see that the vagina is normal.

It's adherent, but it's normal. And what happens when all three bits of the posterior compartment have deep endometriosis? That's what you can see in this example here. You can see this patient has deep endometriosis in the posterior vaginal fornix.

It's fibrosis, retrocervical fibrosis with deep endometriosis within it just there. And you can see the bowel module. And so you can see that both the vagina, the ligaments and the bowel all have deep endometriosis.

And this is palpable on vaginal examination. And gynecologists call this a rectovaginal module. So bowel endometriosis, if you can tell the surgeon about the dimensions, about the angle the bowel's making, the percentage of circumference affected, and therefore whether it might be stenosis, how low it is, if it's intra or retroperitoneal.

If there are more than one lesion, this is very common, either in the same segment or in a different segment. And if there's a rectovaginal module. So you can see that ultrasound can see so much more than at laparoscopy.

That was the normal pelvis. This is the frozen pelvis. And these are video clips from that patient's ultrasound.

You can see that she had a frozen pelvis, that the ovaries, you can see the other way around, that ovaries, that ovary, endometrioma, a little bit of movement, not quite kissing, but also that she had some bowel endometriosis. And that was not visible at diagnostic laparoscopy. Don't forget the anterior compartment.

You can look for nodules of endometriosis in the bladder. You can look for smaller lesions and measure the distance to the ureter. And sometimes you get little nodules in the ligaments that come close to the bladder but not in it, particularly if there's adenomyosis, that causes those little nodules too.

Look for the ureters. They can mainly be externally compressed from all this fibrosis. And here you can see a hemorrhagic cyst, some deep endometriosis in the ligaments.

And this ureter is closely applied and fibrotic to the ovary with a hydro ureter above it. This is a different case, but hydronephrosis occurs in 3% of such patients. So you need to look for it, always look at the kidneys.

Next, we do soft markers, site-specific tenderness and mobility. We go around the entire pelvis, looking for mobility and tenderness and ask. Is it uncomfortable here or here or here? As you go around the uterus, the ovaries, the adnexy, the bowel and the bladder.

So a normal scan looks at these things, but we all should be doing the sliding sign and looking at the posterior compartment and the anterior compartment. I draw my findings because I find it easier to remember what I saw. You can see here that there was deep endometriosis at the torus, a little bit in the posterior fornix.

Both ovaries had endometrium as they were kissing. And there's a little bit of endometriosis in the bowel. And I also draw it transversely and tell the patients the ovaries have come down to the level of the torus with adhesions to each other, to the torus, to the bowel nodule, but that the kidneys are all right.

I would recommend these resources if you want to learn more about how to scan for endometriosis. And this book is really good. I would recommend that you get that.

So I hope that I've shown you that there are lots of appearances of endometriosis on ultrasound. And I'm hoping that one day, the NICE guideline in 2000, and who knows, that they will not just consider an ultrasound, but that this will become the gold standard, a detailed pelvic scan, once everybody knows what endometriosis looks like. Thank you.

Wow, wow, wow. I think we covered the both ends of the non-invasive spectrum pretty thoroughly. That was amazing.

Thank you, Dr. Johnson. I'm glad we got the video to work. I know we're running up against our promised one hour, but I think there are so many good questions here from the question and answer.

And Martin has some questions from the followers on our social channels. I'm just going to jump right into the Q&A with everyone's permission, if we run over by about five or 10 minutes. Is that okay, Dr. Johnson and Dr. Flores? Perfect.

So there's a couple of great questions from the chat, and I just want to jump into them. I'm going to forego all of my personal questions because these are so good. So one of the questions, and we'll do rapid fire, so that way we can speak to both methods, both ultrasound and then non-invasive biomarkers.

How early on do you think your method is able to make a diagnosis? At what stage or before adhesions form? How early can you detect endometriosis, Suzanne? It's very controversial whether you can see superficial endometriosis or not. And that's a part of your question, I think. Mattia Leonardi has published on this with George Condas, and they developed a technique where they put a little bit of fluid into the pouch of Douglas and could then see these peritoneal irregularities.

Certainly, I can also see them if somebody's just ovulated, let's say, and there's a little bit of free fluid, or even if they haven't, you can sometimes see very superficially invasive nodules of endometriosis in the tissues, but it's absolutely not guaranteed. And I think at this stage in the proceedings, particularly when not so very many people are confident to diagnose endometriosis on an ultrasound, is that if an ultrasound is normal, we should not say there is no endometriosis. I think that's crucial.

That problem where people had a scan and people don't look in the posterior fornix, they don't look in the posterior compartment, and so the scan would be called normal, and then the patient would go back to the GP or gynecologist, and they say, well, it's normal, it must be in your head. This has caused so much grief that I think the last thing I want to do is encourage anything like that. You need to start somewhere.

This stuff is visible. I can see in some people superficial endometriosis. I can certainly see nodules of deep endometriosis or an isolated one in a utero-sacral ligament, and you can see all stages from there.

And so it's a question of not what the most expert people can see, but how can we teach this so that everybody is able to at least come up with a strong suspicion that there is endometriosis here? And Valerie, what about microRNAs? How early can you detect the presence of endometriosis using a microRNA? I'm glad you asked that because I realized I did not include the figure from our study. Actually, the great thing about the second trial, which was looking at the 100 women, it looked at women with stage one through four, and it could be diagnosed regardless of stage. And then have you considered whether your method might make a difference with regards to timing it with menstruation or without menstruation? Meaning does your method of diagnosing endometriosis perform better or worse depending on when you do it along the cycle? Dr. Flores, I'll start with you.

With respect to the microRNAs, no, because we look specifically at proliferative and secretory phase, and there was no difference. They were still elevated regardless of the cycle phase. And looking at treatment for, sorry, biomarkers for treatment response for progestin-based therapy, interestingly enough, what I didn't get to talk about for that original trial, looking at PR status and lesions, some women were actually on the same therapy that they were feeling.

So progestin-based therapy at the time of surgery, and then also looking at menstrual cycle phase, it actually did not make a significant difference whether or not they were on medication or not. And then the cycle phase, we're still looking at that just because we had more women on medication than women who were actually cycling on their own. And Suzanne? For me, it makes no difference to me if somebody is having a period at the time of the scan, I'm aware that they will be in pain because whether they have endometriosis or not, a lot of people have period pain.

So I would be even more gentle and make sure the woman's happy to be scanned because not everybody is when they're menstruating. But doing the scan during menstruation can give you extra information that you wouldn't have had otherwise. For instance, if somebody has, let's say, cervical stenosis and has a retrograde menstruation, which can be normal, but it can be as a result of outlet obstruction, let's say, you can sometimes see the retrograde menstruation through the tubes.

You can see the fluid in the pouch of Douglas. That then sometimes allows you to see more adhesions or nodules of endometriosis, but it can help guide you to where the disease is worst and why they're having quite such pain. And also when somebody is menstruating, they've likely ovulated and bled into an existing endometrioma.

And so you can see that, you can see here's the endometrioma and look, this is recent hemorrhage into it. And so explain to the patient why it's quite so excruciating at the moment, but not forgetting that in some people with the worst endometriosis, they have no pain at all, no pain and no tenderness. And that is fascinating.

It's very lucky for them, I often think, but the presence or absence of pain doesn't necessarily help. And I have one more question before I turn it over to Martin. This is a question from Madhuri Salgar.

What in addition to progesterone treatment do we have for endometriosis? And Dr. Flores, the question is directed to you. Do you think these micro RNAs that you're using for diagnostic purposes may also be used as for therapy to treat endometriosis? Yes, that's the goal. So a couple of different goals.

One is assessing treatment response, which are ongoing studies, both in our group and in other groups. So whether or not, let's say someone is on progesterone-based therapy, we can check, we can see the micro RNAs and whether or not they decrease and if that's an indication of treatment response. We also have work looking at micro RNA let7b.

It's not one that I talked about with respect to this presentation, but we have seen that micro RNA let7b is significantly decreased in women with endometriosis and let7b is really involved in blocking inflammation. And so in a murine model of endometriosis, we actually provided essentially the let7b that was missing into these mice and did see decreased lesion size as well as decreased aromatase expression. And ER alpha and beta expression.

Martin, do we have any questions from our social media channels? We do, yeah. Thanks so much, Pietra. And thank you, Valerie and Suzanne.

There's a question from Instagram from someone called Ran2Face who's asked, has there been any economic evaluation of potential savings of these amazing tests that you've described? If we could start with Dr. Flores. I think that's really what we need to start looking at, because the, at least in the US, the healthcare costs does exceed 22 billion, and that's in part due to the fact that one, there's a diagnostic delay, and two, that right now currently people, some people are relying on surgical diagnosis. And so I'm hoping that as we shift more towards this non-invasive diagnosis, and also shift towards earlier diagnosis, that'll hopefully reduce healthcare costs.

And Dr. Johnson? The cost of a scan done well is very, very small, but the cost of a scan done badly, not intentionally badly, but purely because the person hasn't learned how to do this, can be very high. And I think a lot of the eight-year delay is caused by a scan or a CT or an MRI having been misinterpreted, if you like, because of course you can see endometriosis on an MRI as well, but the same thing holds is that a radiologist needs to have been trained in gynae radiology, because otherwise, they may also miss the disease on cross-sectional imaging. And so I think from a cost-effectiveness point of view, it will be a complete no-brainer to train up some gynaecologists who will specialize in ultrasound rather than surgery, let's say, or maybe both, and they would be called sonologists, but also educate all of the sonographers.

And in the UK, we are planning to do that with the British Medical Ultrasound Society, that they are beginning with me and some other people to write a program of training, starting with the sliding sign. A sliding sign is quick and easy to learn, and even just doing that will help to pick up a bunch of women with a frozen pelvis or with fewer adhesions than that, and that will be very cost-effective. But the sonographers have to be allowed enough time to do the scan.

And when I said half an hour or 45 minutes, that includes calling the patient in, getting the bed clean, getting them on it, explaining what you're doing, taking a history, really important, doing the scan, getting them all off again, cleaning the bed, explaining to the patient what you've seen. And all of that simply cannot be done in the 20-minute slot that a lot of sonographers get for an ultrasound. They're being set up to fail in that incident.

So it could be extremely cost-effective, and I'm very positive for a much better future for diagnosing women with endometriosis earlier. I'm so pleased that education's on the agenda for both doctors, sonologists, as well as sonographers here in the UK, and I'm really pleased to hear that it's entering the curriculum. This question's gonna be a person to you, Dr. Johnson, and it's from Rosa in 85 on our Instagram channel.

And she says, can you tell the difference between scar tissue that's formed from previous surgery and deep nodules of endometriosis? And she said they both form from kind of similar processes of fibrosis or scar tissue. Yeah, you can tell the difference. The thing that shows up an active endometriosis lesion with fibrosis around it is what I illustrated as the dark nodule with white around it.

That's endometriosis. Whereas you can see fibrosis from let's say previous operations, but they, and it will be white and fibrotic, but you don't get the dark centers in it at all. So it looks completely different.

Fantastic. Thank you so much. And Martin, if we may, since I know we're running up against people's evening, I have one question that I'd like to ask Dr. Flores that I don't think she touched on her presentation, but really I've been interested and would love to get an answer.

How does the presence or absence of both deep infiltrating endometriosis and endometriomas impact the micro RNAs that are being expressed? Is it dose dependent? If you have this big inflammatory endometrioma, are you gonna get more micro RNAs or are you gonna get different micro RNAs? Or is it kind of all the same? Yes, no endometriosis. Right now, I hate to overgeneralize right now. I would say that it does appear to be about the same, only because in the second trial, looking at a hundred subjects, 50 with endo, 50 without endo, when looking at the stages, there was no significant difference in the micro RNA expression, other than it was obviously increased in women with endometriosis.

My suspicion is that with time, we will see, and with a larger sample size, we probably will see that certain types of endo may have a different micro RNA signature than other types of endometriosis. And then from a tissue perspective, I feel like sometimes the other question is, why not look at tissue? Two reasons. One, it does make it obviously more invasive.

And two, because these micro RNAs are so stable, people have noted that there is a similarity between what's in the tissue versus what's in the serum. And so the serum makes it easier for a non-invasive test. Thanks.

That was perfect. Martin, any final thoughts? I guess it's a question of my own, and for someone whose basic science knowledge is probably on the lower end. My understanding of micro RNAs are that they're post-transcriptional markers that influence protein expression.

Correct, correct. And are they thought to be, are the differences seen in your studies thought to be as a direct result of the disease causing inflammation and changes? Or is the theory that the changes that are seen in these people with varied micro RNA profiles likely to then cause them to go and get endometriosis? That is the million dollar question, Martin. It's a question of the chicken or the egg, because we know that endometriosis, for example, causes inflammation.

Is it the endometriosis itself then that's causing all the inflammation with the altered immune response? Or is it that these micro RNAs were initially aberrant? And since we know some of these micro RNAs also contribute to inflammation, are they what's allowing the disease to progress? I don't think we have an exact answer yet. I think what we are seeing though is some of these other symptoms that didn't necessarily go along with the exclusive pelvic manifestations can be explained by endo having long range effects mediated by the micro RNAs. I know that's not a perfect answer.

I wish I had one, but I think we're still researching that and still trying to understand that a little bit better. Lovely. Thank you.

And I guess the role of ultrasound would come into this fantastically as there've been several studies published from a center I used to work at in University College London where they mapped people with no endometriosis, had acute pelvic hemorrhage, ovarian hemorrhage, and then went on to develop deep infiltrating endometriosis they think is maybe related to the ovarian hemorrhage. And I guess it would be interesting to track the micro RNA profiles as those people perhaps prior to that during and then after. But a nice marriage of the two kind of skill sets that have been presented here today in the translational form of research.

Certainly I've seen that as well and been able to track some women who had an acute hemorrhage and then developed endometriosis, but it's a little bit similar to women who have congenital abnormal uterus and malarian abnormality, which comes first, the chicken or the egg? Does that abnormality causing retrograde menstruation cause endometriosis? Or is there something congenital that has laid down some endometriotic tissue in utero that then causes both the malarian abnormality and the endometriosis? And I think endometriosis is a fascinating subject. And there's clearly a need for a huge amount of research to come. I completely agree.

And I think I'll continue to work doing my little bits. And nowhere near as fantastic and groundbreaking as the two of you here today. But from on behalf of the BSG, I'd like to thank you both for attending and thank you, Pietro.

Pietro, do you have any final words? No, I just want to say that everything that we discussed today will be recorded for posterity. So if you missed it or weren't able to join live, you'll be able to access these recordings both on the BSG website as well as on the fertility and sterility website. And if you have questions, I know Dr. Johnson is on Twitter.

Dr. Johnson, can you share your Twitter handle for people who may want to learn a little bit more about ultrasound? It's at gynecology US. Gynecology is spelled the UK way with AE. So at gynecology US is my Twitter handle.

And Dr. Flores, we got to get you on Twitter. I have Twitter actually. Perfect.

I just pulled it up because I couldn't remember my handle. I'm sorry. It's at VA, V as in Victor, A as in Apple, Flores, F as in Frank, L-O-R-E-S as in Sam and the number seven.

Perfect. And if you missed all of that, you can just PubMed her. There's a lot of work that she's been doing on this topic.

And you can find her contact info there. But like I said, thank you for all of you for tuning in at a late hour in Europe, certainly in the end of a busy clinic day in the United States. And we look forward to seeing you again at hopefully a next joint BSGE in fertility and sterility event.

Thank you so much. Thanks for tuning in.