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Feinstein Institute Researchers Develop Noninvasive Endometriosis Diagnostic Assay

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NEW YORK (GenomeWeb) – Researchers at the Feinstein Institute for Medical Research have developed a noninvasive blood-based assay that uses menstrual effluent samples to determine whether a woman is suffering from endometriosis.

Endometriosis is one of the most frequently diagnosed health disorders in women, with up to 10 percent of reproductive-age women suffering from severe periods and possible infertility. While the disease's cause is unknown, researchers believe that retrograde menstruation may influence growth of endometrial lesions outside the uterus and adhere to the pelvic cavities, causing inflammation and repeated scarring.

Diagnosing individuals with the condition, however, is often delayed and hidden for years, as many women choose not to have the costly laparoscopic surgery needed to detect endometrial lesions. In addition, many asymptomatic patients are only diagnosed with endometriosis during infertility workups.

In a study published in Molecular Medicine earlier this week, Peter Gregersen, its corresponding author and head of the Robert S. Boas Center for Genomics and Human Genomics at the Feinstein Institute, and his colleagues analyzed menstrual effluent (ME) using flow cytometry to isolate stromal fibroblast cells (SFCs), a type of stem cell that helps the uterus regrow its interior lining each month.

"Prior studies, using invasive samples and endometrial biopsies, showed that fibroblast cells had defects in decidualization, a process that allows the uterus to prepare for pregnancy," study coauthor and Feinstein Institute professor Christine Metz said in an interview. "We [therefore] decided to use stromal fibroblast cells in menstrual effluent to see if they showed the same defect."

The Feinstein team recruited patients with endometriosis through the institute's Research OutSmarts Endometriosis (ROSE) study, and control subject from the organization's Genotype and Phenotype registry (GaP), whose database contains genetic information on over 7,000 individuals.

The researchers stimulated the SFCs in controls and subjects with endometriosis (cAMP) at three different times within a two-day period, measuring IGFBP-1 production as a marker of decidualization at each point.

The team then collected RNA and performed RNA-Seq and qPCR analyses to identify additional biomarkers of decidualization and genes differentially expressed by ME-derived SFCs obtained from controls and endometriosis patients.

The researchers noticed highly significant differences in the decidualization response: ME-derived SFCs cultured from endometriosis subjects showed impaired decidualization potential compared to controls.

For genetic markers, the Feinstein group profiled stromal cells obtained from women with endometriosis versus control subjects. They examined ALDH1A1 with qPCR probes and saw that it had reduced expression in SFCs of endometriosis patients, compared to controls. ALDH1A1 is a biomarker normally expressed differentially across the menstrual cycle.

Metz believes that both environment and genotype play a role in modifications that may occur in patients with endometriosis, and noted that a recent meta-analysis of over 170,000 endometriosis patients and almost 200,000 controls revealed 14 genetic regions associated with endometriosis, which may "shine light on the mechanisms of genetic predisposition."

"We believe that one of the major benefits of this study is to open up the possibility of carrying out population-based genotype-phenotype studies that are relevant to better understanding the underlying pathogenesis of endometriosis," the researchers said in their study.

Metz acknowledged that the study's small sample size is a major issue, as the team tested only seven control samples and seven patients surgically diagnosed with endometriosis. To combat the issue, the researchers collected multiple ME samples from each patient during the three days of the menstrual cycle, and over multiple cycles for several subjects. In addition, the team aims to perform the assay on a much larger sample size at the Feinstein Institute as part of the ROSE and GaP genetic registries.

Gregersen also acknowledged that future studies will need to address multiple issues, including but not limited to establishing clinical specificity and sensitivity; determining whether there is heterogeneity to the disease; if the phenotype change occurs after people are treated for endometriosis; and if the phenotypic changes in ME can predict the rate of progression and severity.

"It's been an uphill battle to try and convince people that studying menstrual blood is worth doing," he explained. "There are very few people who have studied menstrual blood in any detail."

Gregersen hypothesized that there are expression phenotypes that will reflect the decidualization defect, but that they will need to be further explored to potentially become part of the diagnostic test.

"[We] suspect that the genetics underlying endometriosis will be quantitative, influencing the degree to which the decidualization occurs, or the degree to which these implanted lesions are dealt with by the immune system," he noted. "Stromal cells have a functional device with endometriosis, but whether that is directly related to [the condition] and how that directly works is not entirely clear."

Metz said that the team's long-term goal is to commercialize the test so that endometriosis can be identified across all of its stages. Her group is applying for further funding and plan to apply for a patent for the decidualization assay later in the year.

Since developing the proof-of-concept test, Gregersen and Metz are now further developing the technology to reduce the wait time for SFC cell growth.

While the test is only at the proof-of-concept stage, Metz said that the cost per test would be relatively inexpensive as long as the clinician is able to collect ME samples, adding that the test's hypothetical cost is cheaper than laparoscopic surgery and easier to perform.

"All you'd need is a blood-collection container able to hold ME, putting it immediately into a solution with antibiotics and antimycotics to prevent contamination, a small isolation step, before running the decidualization of the assay and finally a qPCR assay for validation," she explained.

By using freshly isolated cells from the menstrual blood, the team's assay would be able to run samples almost immediately and reduce turnaround time for patient results. Currently, a diagnosis can take up to 10 years because endometriosis can be confused with other medical conditions. Further, diagnoses may be delayed between five and 10 years because patients try to avoid the current surgical method.

With the assay being developed by the Feinstein team, a diagnosis can be done in less than three days, according to the researchers. "We've developed an assay that can work with 4,000 cells, and isolate just the SFCs that are there, rather than growing them for three weeks," Metz explained. "We can freeze menstrual fluid in time in a way that doesn't kill the cells."

Gregersen added that the assay uses "anywhere from 0.5 to 5 ml of the [participants'] menstrual effluent," and said that the commercialized assay will either involve gene expression or proteomics, as "we have a lot of ideas, and the early signaling pathways for decidualization may be captured by using a proteomic approach."

While the Feinstein researchers Institute continue work on a miniaturized assay for endometriosis in the clinical space, other groups have also been developing diagnostic tests for endometriosis in the last few years. Salt Lake City-based Predictive Technologies is commercializing an assay to search for genetic biomarkers that the firm purports may detect the presence of endometrial tissue. Unlike the Feinstein team, the firm uses blood and saliva samples in its PCR-based diagnostic and prognostic assays.

In addition, MDNA Life Sciences formed a collaboration last year with the University of Oxford to further develop and validate its mitochondrial DNA-based blood test for endometriosis. The noninvasive liquid biopsy test is planned for launch later this year.

Jill Rabin, a professor of obstetrics and gynecology at Hofstra/Northwell, said that future studies would need to focus on uncultured ME cells, and control groups would need laparoscopically confirmed absence of endometriosis.

"If you use menstrual effluent, you're basically getting to the root of the problem, and from a proteomic and molecular standpoint, you may be able to interrogate more markers," Rabin emphasized. "[Menstrual effluent] may have certain markers that are expressed more in the source tissue, rather than in systemic body fluid."

The Pelvic Rehabilitation Medicine, a New York-based medical center, plans on partnering with the Feinstein Institute to actively recruit patients for future studies to assess the potential impact the test might have on patients in the long term. Its medical director, Gautam Shrikhande said that the Feinstein team should examine important patient-related issues, including the severity of endometriosis cases and the patients' clinical backgrounds He agreed that the test could help inform the patient's relatives of their likelihoods of developing the disease.

"If [the patient] can tell their sister, daughter, or other female relatives, it can have a significant impact on the suffering generations to come," he noted.