Stromal Secrets

When patients present with pelvic pain or infertility, it’s not often that a doctor’s first thought is endometriosis. It can be even more difficult to have such symptoms taken seriously when the level of pain seems so disproportionate to the disease – and when the gold standard for diagnosis is laparoscopy or uterine biopsy, many physicians hesitate to suggest such invasive interventions for what is frequently perceived as a minor issue. But endometriosis is, in fact, anything but – and, with menstrual effluent providing a potential new, noninvasive approach to testing, patients with the condition may soon receive the diagnosis and treatment they so desperately seek.

From HLA to WNT4

Peter Gregersen has been interested in genetics for a long time. “I originally trained as a rheumatologist in New York, studying at Columbia and New York University, and did my early work cloning human leukocyte antigen (HLA) genes involved in rheumatoid arthritis,” he says. That work took place in the early 1980s, just at the beginning of the cloning revolution. Gregersen and his colleagues cloned the major HLA genes for rheumatoid arthritis and developed a hypothesis – called the “shared epitope hypothesis” – for how those genes worked. From there, he thought, “Well, we’ve got big HLA effects – maybe there’s something going on in T cells?” He spent some time working on T cell repertoires to explore that possibility further – “but by the 1990s,” he says, “I was back to my first love: genetics.”

That’s when he formed the Northern Rheumatoid Arthritis Consortium – the first consortium for genetic analysis of the disease – and, since then, they have produced 100 hits and become an international group, the Rheumatoid Arthritis Consortium International. “As you can see, most of my life has been focused on mapping genes and autoimmunity, and – more recently – on analyzing the functions of those genes.”

Of course, there are many complexities: most diseases have lots of genes involved, each with a very low individual effect. A lot of those involved in autoimmunity seem to impact T cells, B cells, and other immune functions. Over the last five years, Gregersen and his research colleagues have focused on studying those same variants in individuals who neither have the autoimmune disease of interest nor are on medications, so that they can really tease apart the functions of those variants in the absence of all those complications. In recent years, they have published heavily on T and B cell genes – the ones that regulate how quickly a B cell will respond to a stimulus, or how high T cells will jump if you stimulate them in standard ways. “A lot of these genes can influence endophenotypes, so I think the current focus is on trying to understand endophenotypes in the context of these risk alleles,” says Gregersen. “It’s something I have done with lupus, arthritis, multiple sclerosis, myasthenia gravis, and others over the years – but then, about five years ago, some colleagues who suffer from endometriosis came to talk to me about it.

“I didn’t know anything about endometriosis at the time – but I found some interesting genetic research on loci associated with low odds ratios. WNT4, the top endometriosis gene, is directly involved in the decidual response, which is disrupted in the condition.”

Defects in decidualization

Rheumatoid arthritis is a common disorder that has captured many researchers’ attention. Endometriosis is even more common – but relatively few laboratories worldwide are seriously working on it.  A major focus of the work so far has been on defining the genetics, as well as on trying to stage the disorder, because it features variable types and degrees of severity: there are lesions scattered around the peritoneum that bleed and cycle with the periods; there are chocolate cysts that engulf the ovary and impair ovulation; and there is invasive, often debilitating lower pelvic floor disease (perforation of the vaginal wall, bowel, or bladder). Having spoken to a lot of people with endometriosis, Gregersen says he has learned that the level of pain does not always follow the severity of the disease – but we don’t yet know why.

Aside from not really understanding how endometriosis works, there are two main issues with the condition. One is the delay in diagnosis – up to a decade! A typical story would be a high school or college-aged woman coming to a school nurse or doctor with these symptoms; they don’t get recognized as being endometriosis – “Oh, just suck it up; this is what menstruation is all about; we’ll put you on a nonsteroidal anti-inflammatory or ‘the pill’” – which can sometimes be effective, but many people ultimately need surgery. Worldwide, surgeries are performed by a few well-trained surgeons. Although there haven’t been controlled trials, it seems likely that stem cells drive these lesions, so removing the entire lesion – including the stem cells – is extremely important. If the surgeon simply cauterizes the lesions, they will return. In fact, it’s quite common for women to have had multiple operations, because they experience a temporary improvement after each one, but then have to go back in because the lesions weren’t fully removed.

Many endometriosis patients come to the attention of physicians because they have pain (though they are often dismissed because the intensity of the pain seems so unlikely) – but with others, the second issue arises: infertility. “About 30 to 40 percent of people who present with female infertility have endometriosis, and if you treat that, you often restore fertility – possibly because the disease interferes with ovarian function or with decidualization (which is important for implantation),” Gregersen theorizes. “Unfortunately, the only way to conclusively diagnose endometriosis is by laparoscopy to see the lesions; there are no noninvasive tests. That’s what initially made me think we needed to stop focusing on the immune system or the lesions themselves; instead, we needed to look at the source of the cells causing the lesions – the menstrual blood. Almost all women experience retrograde menstruation at every cycle, and yet only five to 10 percent get endometriosis.” Why is that? One possibility is that there are cell differences that cause the condition – and that’s something that can be studied by collecting menstrual blood from women with endometriosis.

Gregersen began working with a reproductive biologist, Christine Metz, at the Feinstein Institute about five years ago. This has evolved into a full-scale collaboration on endometriosis. “The first thing we learned was that, if you put menstrual blood into culture, stromal cells grow out of it like crazy. These are fibroblast cells that have some pluripotent ability. They grow very rapidly, and they exhibit decidualization defects that can be observed on uterine biopsies of people who should otherwise be in the late luteal phase.”

Gregersen and Metz decided to see whether these cells could be induced to decidualize. Standard approaches to decidualization use cyclic AMP and combinations of progesterone and estrogen, so his group took that approach and, lo and behold, stromal cells from people with endoscopically confirmed endometriosis proved not to decidualize well, whereas normal cells did. “Great news,” says Gregersen, “because what we need is a biologically relevant diagnostic, and these stromal cells may be exactly that. At the moment, we still need a visual examination to document endometriosis, so women who present with symptoms of the condition must undergo laparoscopy. It’s our hope that, eventually, we can examine the stromal cells of such patients to determine whether or not they are likely to need a more invasive procedure.”

Diversity and heterogeneity

Obviously, this work is still in its early stages. One criticism the group has heard heard of their method is that they haven’t staged the disease. “We’re going to get there!” says Gregersen. “First, we need to apply the test to a large number of people with well-staged endometriosis and see if it correlates with severity. We have yet to establish the test’s sensitivity and specificity. And we’re currently collecting menstrual effluent from people who present with infertility to see whether or not it might be useful in that setting. Finally, we’d like to establish how the test results change after a patient’s lesions are removed – is it a genetic defect that remains, or is it (as previous data suggest) a phenotypic outcome of the lesions that resolves?”

These kinds of questions can be addressed using a genetically characterized normal control registry that Gregersen set up about 10 years ago. It contains data from about 5,000 people with no symptoms of endometriosis – but some of them carry risk genes like WNT4. He and his colleagues are now asking them for menstrual samples so that they can see whether or not those who carry the WNT4 risk haplotype (which includes an abnormality in the gene’s estrogen-binding site) also exhibit decidualization changes. “Work like this might help us understand the biology of decidualization without having to perform uterine biopsies to gather data. We can investigate genetic variation in populations and ask specific phenotypic questions – or vice versa. I suspect that there are multiple paths to a decidualization defect; GWAS has revealed a number of genes in the pathway. There is clearly heterogeneity in terms of severity (for instance, WNT4 is more associated with more advanced pelvic floor disease), but the factors influencing it are not yet clear. There’s a broad diversity of endometriosis phenotypes, so I expect we’ll see similar diversity in our molecular

A need for further study

Conditions like endometriosis are often understudied. Gregersen says, “This is partly because there’s not much funding available and partly because – in my opinion – women’s diseases in general are underappreciated and underdiagnosed. The awareness of endometriosis has been very low until recently; after all, I myself am a well-trained physician who attended a highly ranked medical school, and yet I was completely unaware of it until it was called to my attention.”

He acknowledges that his approach is also unusual. “This field is largely driven by surgeons, but even those I know and respect were skeptical of working on menstrual blood. ‘Why don’t you work on understanding the lesions? Get the cells out, find out what types are involved and what cytokines they are making…’ It makes sense, of course, but it requires getting surgical specimens. And the lesions are incredibly diverse, so it’s hard to decide which are truly representative.”

The heritability of the condition is estimated at about 50 percent, and beautiful work has been done to identify at least 14 regions associated with endometriosis using large population association studies. Gregersen suspects that, as with autoimmune disorders, there will be genetic subsets of the disease, and there will be families with endometriosis that probably have much more highly penetrant genes in addition to those that have been mapped by GWAS. Those families may be incredibly important to find, even if uncommon, because they may yield insight into disease pathogenesis. “To find out more, we’ll need to recruit large families, perform genetic testing, and see how the results correlate with disease phenotypes,” he says.

Developing a diagnostic

A test based on the biology of stromal cells may well one day become a diagnostic that could help drive the selection of therapy. Some patients respond quite well to progesterone therapy. Others need more intensive intervention; for instance, doctors may put some patients into menopause or perform surgery to remove the lesions or even the entire uterus. “Of course, these are treatments we’d like to avoid if at all possible – so if we can identify patients who might succeed with less aggressive therapy, we can ensure we’re adhering to the basic principle of medicine: first, do no harm.”

Gregersen also thinks that testing stromal cells in menstrual blood has applications beyond just endometriosis – for instance, perhaps in adenomyosis or fibroid tumors. “We have considered developing new collection methods for menstrual blood, such as single-use cups or specialized tampons, but for the moment, patients seem most comfortable with the reusable cup. Some patients are hesitant to perform the collections (and some physicians are hesitant to ask their patients to do that!), but with problems as life-affecting as endometriosis or infertility, many women are happy to undertake the task if it carries the potential for answers.”

The next step for Gregersen and Metz’s research program is to miniaturize this process and make it more efficient. “We can now grow cells from fresh menstrual effluent for 48 hours and get a decidualization assay from 3,000 cells (or even fewer) – it’s amazing! I think we can miniaturize that assay so that it can be performed on a 96-well plate and still take only 48 hours to complete. We are also looking into gene expression data. The cells actually appear to be different at the single-cell level, but it’s work we’ve only done on cultured cells thus far; fresh effluent is our next step. We may also try freezing samples (which doesn’t change cell distribution) for later culturing and analysis. And, finally, we’re collaborating with colleagues at Cold Spring Harbor Laboratories to look at possible proteomic changes. Very little is known about the signaling pathways in terms of possible phosphorylation changes after decidualization; my guess is there may be defects in that pathway that we could capture just by stimulating the cells and looking at the phosphorylated proteome. That way, we can see where the potential defects lie.”

Ultimately, Gregersen says, he’d like to be better able to help at-risk populations who present to infertility or pelvic pain clinics. The prevalence of endometriosis in those patients is likely to be quite high, and for them to be able to get the help they need, doctors and researchers need to pay this condition the attention it deserves.