NEW YORK – A French team led by investigators at Institut Cochin in Paris has received €1.5 million ($1.7 million) in EU funding to develop biomarkers for endometriosis, a condition where tissue similar to the lining of a woman's uterus grows outside of it.
The five-year project, called MensEndoDiag, is using menstrual blood as a sample type. According to Ludivine Doridot, a researcher at Institut Cochin and an associate professor at Université Paris Cité, menstrual blood has been overlooked as a sample type, even though it is highly accessible for molecular testing.
"It's been overlooked for two reasons," said Doridot. "The first is a social barrier, as some people believe it is disgusting." The second is that feminine hygiene products until rather recently did not allow women to easily collect their menstrual blood. The increased use of reusable menstrual cups has changed that, and to the benefit of researchers interested in women's health.
"It isn't a hard sample to work with," said Doridot. "There are also a lot of viable cells. When you ask women, they are happy to give it to science."
MensEndoDiag will conduct several types of biomarker discovery, Doridot noted. The first is to combine single-cell transcriptomics data obtained via next-generation sequencing with protein biomarker data to discover markers that could be used to diagnose endometriosis. The researchers will recruit patients representing different endometriosis subtypes — superficial, ovarian, and deep-infiltrating — and also hope to define new subtypes using machine learning tools.
The researchers will perform library construction and RNA-seq using 10x Genomics and Illumina platforms housed at the Institut Cochin's genomics facility, Doridot said.
The team is also considering using LegendPlex assays from San Diego-based BioLegend to measure a custom panel of protein markers, Doridot said. LegendPlex is a bead-based immunoassay that can be read via flow cytometry in a 96-well plate format.
MensEndoDiag's discovery cohort will involve 64 patients, both cases and controls, sourced from Hôpital Cochin. Once the researchers define a marker set, they intend to validate them in menstrual fluid samples obtained from a cohort of 250 women. This will take time though, as many women use birth control pills, which makes it more difficult to find patients with endometriosis who can provide menstrual blood samples. Just assembling the discovery cohort might take a year, said Doridot.
Doridot would also like to develop a panel of protein and RNA markers that could be used to subtype endometriosis patients. "I hope we will be able to develop something that is a combination of both," she said. "You can easily associate an antibody with a nucleic acid tag, and you can assess both protein and RNA at the same time."
The researchers will also culture menstrual fluid-derived organoids with and without immune cells to assess functional changes and test out new immunomodulatory treatments. In 2020, Doridot and fellow researchers published a paper in Cell Reports that discussed the responses of mouse endometriotic cells to macrophages primed with low doses of lipopolysaccharides, and noted that the researchers may perform a similar study using the cultured human cells.
Finally, the investigators also aim to identify potential biomarkers in menstrual blood to predict a patient's response to surgery or in vitro fertilization treatments.
These markers, she noted, could be the same as the ones used to diagnose and subtype endometriosis. "We don't know yet, but we will see if there is any commonality between the diagnostic and prognostic biomarkers," said Doridot.
Several companies have developed or are in the process of developing molecular tests for endometriosis that rely on menstrual blood as a sample type. One is NextGen Jane, an Oakland, California-based outfit that in February 2022 received $1.5 million from the US National Institutes of Health to continue work on a test for endometriosis that would be run on menstrual blood.
In addition, San Diego-based Xosomix was awarded $318,000 from the National Institutes of Health last fall to develop a menstrual blood-based endometriosis test that relies on multiplexed quantitative proteomics. Also last year, Innovative Health Diagnostics and AIMA Laboratories said they were developing an at-home test for endometriosis that will rely on a microRNA biomarker panel.
NextGen Jane CEO Ridhi Tariyal said in an email that menstrual fluid provides a "unique mixture of cell types" including immune cells not found in any other sample type, as menstruation is a hormonally driven inflammatory response. Such cells "coordinate the process of menstruation and play important roles in the response to bacteria and pathogens found in the vaginal and uterine environments," she added.
Tariyal agreed with Doridot that this sample type has been overlooked for biomarker discovery and diagnostic development because of the aforementioned social stigma.
"In our early days we encountered skepticism around whether labs would want to process menstrual material," said Tariyal. "This response was surprising given how other exfoliated materials, such as stool samples, have seen regular adoption for both microbiome testing and colorectal cancer screening."
Despite this, clinical study participants often send NextGen Jane multiple tampons for analysis, Tariyal said. "We see this as a compelling affirmation of integrating into normal routines for sample collection and creating an elegant sample collection mechanism," she said.
Tariyal pointed out that menstrual fluid also presents some challenges as a diagnostic sample type. For instance, many of the cells in menstrual fluid are dead or in the process of dying, making it more difficult to isolate a gene expression signature. And since menstruation is an immune response, the complex signals detected in fluid might mask a particular disease. The content of menstrual fluid also changes from day to day, and can "vary dramatically" from patient to patient.
"Normalizing for this level of heterogeneity requires both knowledge of the biological changes within this sample type, as well as bioinformatic approaches that can account for the variability and stratify these dynamic signals into meaningful data," said Tariyal. She acknowledged that creating the internal processes to analyze such data took the most time in the development of NextGen Jane's platform.
"But once you have a robust process in place," she said, "the disease insights can be profound."