Katie Burns, PhD - Can White Blood Cells from Menstrual Fluid Diagnose your Endometriosis?

Thank you everybody. Can you hear me? Yes. Great job. Okay. Thank you everybody for being here. Where's the pointer over here? They're making me move and walk already, which is good because I almost fell off the stage yesterday or the day before. No? Yes? Higher. It's okay, Sarper. It's okay. So they gave me 10 minutes to talk, so I have to talk fast. Okay? So we're just going to talk about the immune system. The normal immune system, when it normally function, is great. As I mentioned earlier, it helps you. It's wonderful. And so it protects you from these invading pathogens like bacteria, fungus, parasites. However, when it's not quite working right and it fails, it doesn't have the ability to protect you and it can even attack your own body. And so I think some of that is what we've talked about today and how sometimes we can have what is considered an exaggerated response to the immune system.

And that may be some of what's happening with endometriosis. So the immune system is complex. And I have a friend who is absolutely brilliant and she is an immunologist. And I tell her that she's at the top of the scientific food chain because there are so many cells and different things in blood that we don't even completely understand. But we're going to try to understand a little bit of it today because there's called the innate immune system and the adaptive immune system. And there are different cell types in each of these. And how I like to think about it is if you're in a car accident and you need to be stabilized, you need to get to the hospital, those are your innate immune system cells. However, our adaptive immune system cells are the ones that make the antibodies. They're the ones that put us back together to keep us more healthy.

And I consider those like our trauma surgeons. So cells of our immune system also provide the surveillance to remove dead and dying cells, and that's important to menstruation. And these are a part of the innate immune system. We all know menstruation, but this is one of my favorite images and I'd like to show this however they did this is just phenomenal because you have both the ovulation, the ovarian cycle, as well as the uterine cycle. And there are so many changes that happen in both the ovary and the uterus. But the important point of this is menstruation is incredibly inflammatory. So there's progesterone withdrawal, which leads to release of chemokines or molecules from your immune cells that recruit other immune cells into the environment. The environment is pro- inflammatory. And as I mentioned earlier this morning, it really leads to resolution of what is actually happening in the shedding of that tissue here during menstruation.

So the endometrium is broken down, it's released, and after menstruation, immune cells remove some of that cellular debris and they help with remodeling that gets ready for the next cycle. One of the things that I noticed initially and couldn't quite understand about people looking at menstrual fluid is that they miss neutrophils. So neutrophils are one of your first responding cell types. They are the most abundant white blood cell in your peripheral blood, but we weren't seeing them in some of the studies. So I said," Let's look more closely at menstrual fluid and see what's happening. "So we used menstrual fluid as an investigative tool. This is our study design where we had endometriosis patients and healthy controls. We included women who were 18 to 45 with regular menses and women with endometriosis had to have laparoscopically proven disease. And we had a number of exclusions to make sure that we really dialed in on our study and focused on what we were doing.

We also learned that having an IUD in place with the use of a diva cup can actually pull out the IUD and you don't want to do that. So that's one of our exclusion criteria right now. So with that, what we use is the diva cup and we collect menstrual fluid on days one and days two of menstruation. We have patients or participants take the menstrual blood from the diva cup, put it into a tube, and then we bring it to the lab, we transfer it to the lab. And this is an example of what we get in the lab, very minimal volume because we often think we bleed a lot. It's not necessarily as much as we think it is, but it does sometimes fill up a tube. So this is an example. We then filter that through a number of different meshes, strainers, filters.

This one is actually a healthy person because it's beautiful. And what you can see is this very clear piece that corresponds to like your plasma or your serum. If you are doing a blood test and they're looking for your blood test. Down at the very bottom are your red blood cells and then there's a layer of different kind of lighter colored cells. Those are your white blood cells. So what we do from here then is to make sure we maintain the fidelity of these cells and to treat them very gently. We do a series of what are called percol gradients or centrifugations. And from there, we actually then can use the menstrual fluid by taking a piece of this and looking at what proteins are in there. We take some of these cells to look at what each single cell might be doing. We also then look by spectroflow cytometry, which is a very specific type of flow cytometry.

It has the ability to look at what's on the outside of the cell surface of these cells so that we can know what the cells could potentially be doing. And then we just looked at them. People hadn't looked at them. So let me show you what this looks like. Okay. This is menstrual fluid without the blood, the red blood cells. It's beautiful, isn't it? So if you look at this, this is a neutrophil.

This is a macrophage. They're bigger. The macrophages are your ones that consider gobbling up things. They eat things. And then you have what are called your eosinophils. And we actually were surprised to find eosinophils in our menstrual fluid because we thought this was only associated with asthma, or not asthma, with responses such as allergies, but actually they play a role in menstrual fluid. We also find then your adaptive immune cells, which are your BNT cells. So if we look at this more closely in control healthy women, you see that these neutrophils have two to three lobes in them and that there are macrophages present, but they have very little activation. You don't see that they're foamy and you'll see that in the next image. But occasionally in some of these neutrophils, we do see more than two to three lobes, and those are considered hypersegmented.

So we went into endometriosis, completely different. And these cells are very different. And so what we're in the process of doing is studying what these cells are and why they're so different because these nuclei are hypersegmented, which means that they have multiple lobes, three to five lobes. And another thing that we're studying, we don't know what they do, but they have multiple vacuules that are found within there. We also see that the macrophages are bigger and more foamy. And one of the places we find foby macrophages is in atherosclerosis and in these inflammatory environments. So is that a potential link to why women with endometriosis have increased cardiovascular issues? We don't know research that we need to do. Okay? And so from there then we got some peritoneal fluid samples from the operating room, and we actually find that those same hypersegmented neutrophils are also in the peritoneal cavity.

And this could lead to some of our other work where we're looking at how these cells could contribute to the development of endometriosis.

This is an example of what we do to get the flow cytometry to see what surface markers these cells are making, but the take-home message here is if we look at the total number of neutrophils that is in the menstrual fluid, there are women that are healthy versus women that have endometriosis, it's the same. However, if we look at those more specific types of cells, the ugly cells, the angry cells, we say that our neutrophils are angry. They're actually aged neutrophils and they have these very specific cell surface markers that allow them to be aged. We find that they're not necessarily dying and that they have ... Whoops. They have the ability to be pro- angiogenic. And angiogenic is a big word for they can help form blood vessels. And so the idea is, can they help form the blood vessels that feed the lesions?

And so there I got my note that I'm running close to time. So this is my last plug. We want your menstrual fluid because you are one in 10. So if anybody's interested in being part of the study, let us know. It's always wonderful to capture, to see if we can add to our study and add to our ability to make sure that we can study this across the age span of women. Right now, you're ready to go. Right now, we actually have looked at women that are on hormones and they have the same neutrophil angry profile. So we have a patent in and we're hoping that we can get funding for this and move this forward. So if any of you know any rich people, come talk to me and let me know. Okay, thank you.