Validation of a Saliva Micro–RNA Signature for Endometriosis, 2025, Bendifallah et al.

[SNT Gatchaman]

Validation of a Saliva Micro–RNA Signature for Endometriosis

Spoiler: Authors

Sofiane Bendifallah; Horace Roman; Stéphane Suisse; Andrew Spiers; Erick Petit; Léa Delbos; Yohann Dabi; Cyril Touboul; Thomas Dennis; Benjamin Merlot; Eric Sauvanet; Raffaèle Fauvet; Estelle Jamard; Hennetier Clotilde; Perrin Morgane; Benjamin Fedida; Krystel Nyangoh; Vincent Lavoué; Claire-Marie Roger; Nicolas Lucas; Thomas Darnaud; Anne-Sophie Boudy; Ludivine Genre; Pierre Leguevaque; Cherif Akldios; Mikhael Benjoar; Elodie Chantalat; Yann Tanguy Le Gac; Mathieu Poilblanc; Pascal Rousset; Hervé Fernandez; Francois Golfier; Philippe Descamps

BACKGROUND
Diagnosis of endometriosis is a challenge. The recent development of a saliva-based micro–ribonucleic acid (miRNA) signature for the diagnosis of endometriosis may enable a timelier and less invasive approach, but this requires external validation.

METHODS
The prospective, multicenter validation of the salivary miRNA signature of endometriosis (ENDOmiRNA) study aimed to assess the diagnostic accuracy, validate the biological reproducibility, and evaluate the clinical utility of a saliva miRNA signature of endometriosis. The study population comprised patients 18 to 43 years of age with signs and symptoms suggestive of endometriosis, who were recruited from diverse medical settings. Patients received a diagnosis of endometriosis by imaging, laparoscopic procedure, or both. All patients who were determined to not have endometriosis were classified as controls (and all underwent laparoscopy). Assessment of endometriosis status based on the saliva miRNA signature was established blinded to patients’ endometriosis status, as determined by imaging and/or laparoscopy and/or histology.

RESULTS
The external validation population was composed of 971 patients, including patients from a prior interim analysis, with an overall endometriosis prevalence of 77%. The saliva miRNA signature had an accuracy (defined as the probability of correct classification for both positive and negative results) of 96.6% (95% confidence interval [CI], 95.2 to 97.6%), a sensitivity of 97.3% (95% CI, 96.4 to 98.0%), a specificity of 94.1% (95% CI, 91.0 to 96.4%), a positive predictive value of 98.2% (95% CI, 97.3 to 98.9%), a negative predictive value of 91.3% (95% CI, 88.3 to 93.4%), a positive likelihood ratio of 16.6 (95% CI, 10.8 to 26.9), and a negative likelihood ratio of 0.03 (95% CI, 0.02 to 0.04). Among patients with surgical confirmation of the diagnosis, misclassification, underestimation, and overestimation rates were 4.6%, 2.4%, and 2.2%, respectively, for the saliva miRNA signature and 27.2%, 15.1%, and 12.2%, respectively, for imaging (either transvaginal ultrasound, magnetic resonance imaging, or both).

CONCLUSIONS
This prospective, multicenter external validation study demonstrated the accurate performance of a saliva-based miRNA signature for the diagnosis of endometriosis in this cohort. (Funded by Ziwig; ClinicalTrials.gov number, NCT05244668.)

Web | PDF | NEJM Evidence | Paywall

 

[ScoutB]

Woah. Why would endometriosis even have a microRNA signature in saliva?

 

[Hutan]

That's sounding pretty exciting from the abstract.

The saliva test appears to have done substantially better at predicting endometriosis (as identified in surgery) than imaging. I wonder how many participants had both imaging and surgical confirmation?

Here's a 2022 open access paper by the same team
MicroRNome analysis generates a blood-based signature for endometriosis
That blood-based study reported

Among the 86 miRNAs composing the blood signature, 10 have the greatest potential value: namely, miRNAs 124-3p, 6509-5p, 548l, 26a-2-3p, 3622a-3p, 3168, 29b-1-5p, 30e-3p, 3124-5p, 4511. The diagnostic importance of the miRNAs is reported in Fig. 2. Among these 10 miRNAs, one (miRNA124-3p) has been previously reported in the setting of endometriosis.

 

[SNT Gatchaman]

Here's a 2022 open access paper by the same team

And the other one —

Salivary MicroRNA Signature for Diagnosis of Endometriosis (2022)

Spoiler: Authors

Bendifallah, Sofiane; Suisse, Stéphane; Puchar, Anne; Delbos, Léa; Poilblanc, Mathieu; Descamps, Philippe; Golfier, Francois; Jornea, Ludmila; Bouteiller, Delphine; Touboul, Cyril; Dabi, Yohann; Daraï, Emile

BACKGROUND
Endometriosis diagnosis constitutes a considerable economic burden for the healthcare system with diagnostic tools often inconclusive with insufficient accuracy. We sought to analyze the human miRNAome to define a saliva-based diagnostic miRNA signature for endometriosis.

METHODS
We performed a prospective ENDO-miRNA study involving 200 saliva samples obtained from 200 women with chronic pelvic pain suggestive of endometriosis collected between January and June 2021. The study consisted of two parts: (i) identification of a biomarker based on genome-wide miRNA expression profiling by small RNA sequencing using next-generation sequencing (NGS) and (ii) development of a saliva-based miRNA diagnostic signature according to expression and accuracy profiling using a Random Forest algorithm.

RESULTS
Among the 200 patients, 76.5% (n = 153) were diagnosed with endometriosis and 23.5% (n = 47) without (controls). Small RNA-seq of 200 saliva samples yielded ~4642 M raw sequencing reads (from ~13.7 M to ~39.3 M reads/sample). Quantification of the filtered reads and identification of known miRNAs yielded ~190 M sequences that were mapped to 2561 known miRNAs. Of the 2561 known miRNAs, the feature selection with Random Forest algorithm generated after internally cross validation a saliva signature of endometriosis composed of 109 miRNAs. The respective sensitivity, specificity, and AUC for the diagnostic miRNA signature were 96.7%, 100%, and 98.3%.

CONCLUSIONS
The ENDO-miRNA study is the first prospective study to report a saliva-based diagnostic miRNA signature for endometriosis. This could contribute to improving early diagnosis by means of a non-invasive tool easily available in any healthcare system.

Web | PDF | Journal of Clinical Medicine | Open Access

 

[SNT Gatchaman]

Multiple factors, including the absence of pathognomonic symptoms, a lack of awareness among both practitioners and patients, the low accuracy of diagnostic tools, and the limited value of imaging techniques, especially for superficial peritoneal endometriosis, contribute to the difficulty of diagnosis. These same challenges often result in a lengthy diagnostic delay: across health care systems worldwide, it takes, on average, 7 years from symptom onset to the diagnosis of endometriosis, resulting in prolonged pain, impaired fertility, psychological stress, and an overall reduced quality of life for patients.

a Cochrane review of the diagnostic accuracy of any combination of noninvasive tests for pelvic endometriosis compared with surgical diagnosis stated: “None of the biomarkers evaluated could be evaluated in a meaningful way and there was insufficient or poor-quality evidence.”

(Huh. Some other Cochrane organisation I guess.)

We now present the full results of the multicenter ENDOmiRNA study, including those of the patients previously described in the interim analysis. 14 The aim of this prospective external validation cohort study was to assess the diagnostic accuracy of the saliva miRNA signature, validate the biological reproducibility of miRNA quantification (temporal and geographic), and evaluate clinical utility in a real-life setting with a prospective comparison of the saliva miRNA signature with routine imaging by transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI).

Enrolled patients underwent either a laparoscopic procedure, an imaging examination, or both. All patients in the control group underwent laparoscopy. Among the patients diagnosed with endometriosis who did not have a laparoscopic evaluation, all patients had an MRI or ultrasound examination showing endometrioma and/or deep endometriosis with colorectal involvement, which was confirmed by a multidisciplinary committee using established criteria.

For patients who underwent surgery, diagnosis was confirmed by laparoscopic visualization and/or histology.

The primary outcome measure of the saliva miRNA signature (previously published random forest model) was analyzed blinded to both the imaging examination and surgical findings.

Samples were indexed in batches with a targeted sequencing depth of 16 to 20 million reads per sample. Sequencing was performed using 75-base single-end reads.

The population below is women 18-43 with chronic pelvic pain, meeting clinical criteria for endometriosis suspicion.

With an overall population prevalence of 77%, the saliva miRNA signature, composed of 109 miRNAs, demonstrated an accuracy of 96.6% (95% CI, 95.2 to 97.6%), a sensitivity of 97.3% (95% CI, 96.4 to 98.0%), a specificity of 94.1% (95% CI, 91.0 to 96.4%), a PPV of 98.2% (95% CI, 97.3 to 98.9%), an NPV of 91.3% (95% CI, 88.3 to 93.4%), a positive likelihood ratio of 16.6 (95% CI, 10.8 to 26.9) and a negative likelihood ratio of 0.03 (95% CI, 0.02 to 0.04).

Maybe another round of spitting in a tube could be in order for a cohort of well-characterised ME patients?

 

[SNT Gatchaman]

Woah. Why would endometriosis even have a microRNA signature in saliva?

From Salivary MicroRNA Signature for Diagnosis of Endometriosis (2022) —

Several studies have reported aberrant expression of miRNAs in affected tissues or peripheral blood samples of patients with endometriosis

To date, Moustafa et al. is the only team to have attempted to build a blood-based miRNA diagnostic signature for endometriosis composed of six miRNAs based on Random Forest analysis. In agreement with previous studies, it would appear illusory that so few miRNAs could reflect the diversity of a multifactorial disorder such as endometriosis, which involves multiple and poorly known signaling pathways.

We hypothesized that a saliva-based miRNA signature for endometriosis would be a low-cost and scalable method allowing samples to be collected anywhere by anyone. The tool would thus be available for underprivileged populations unlike methods based on blood samples, which are blood-volume and temperature dependent, imposing complex logistics of collecting peripheral blood and transporting it to a laboratory for analysis.

Saliva is an increasingly attractive body fluid in the search for disease biomarkers. miRNAs exhibit remarkable stability in severe conditions, such as extended storage. Zheng et al. demonstrated that saliva is not affected by coagulation that could induce a release of miRNAs. This is especially crucial as many studies evaluating miRNA expression in endometriosis have been performed on serum.

Among the 109 mi-RNA of the signature, 29 (27%) are associated with the main signaling pathways of endometriosis: PI3K/Akt, PTEN, Wnt/ β -catenin, HIF1α/NF κB, and YAP/TAZ/EGFR.

 

[ScoutB]

Thanks for posting all the snippets, that’s fascinating.

(Huh. Some other Cochrane organisation I guess.)

Maybe another round of spitting in a tube could be in order for a cohort of well-characterised ME patients?

Yea, seems like it should be relatively cost effective at least. It’s midnight here but now I want to read up on miRNA in other diseases.

 

[Jonathan Edwards]

I have been sceptical about the power of miRNA analysis to help with ME/CFS. I mentioned this to my pain genetics colleagues who have an interest in long non-coding RNAs and they agreed. But I wonder if we should discuss miRNAs in a bit more detail on a thread.

I can see that endometriosis cells and nearby peritoneal cells might chuck out miRNAs of a particular pattern just through random leakage, in the way that prostate specific antigen leaks out of prostate cancer cells.

What I find odd is that they should need to study 109 miRNAs to show the pattern. For most diseases where biomarkers make use of combinations of molecules it is 2, 3 or maybe 4. Presumably the 109 just reflects throwing in anything that adds a little bit more discrimination but it remains puzzling.

My main puzzlement about miRNAs is that I find it hard to see how they could be much use as deliberate biological signals, perhaps passed from one cell to another in extracellular vesicles, as is claimed. If a cell threw out vesicles then why should they reach any other particular cell rather than just got gobbled up by spleen. On the other hand if these are non-specific danger signals or possibly specific signals for lack of something specific like insulin or blood flow then maybe that would not be a worry, if specific vesicle-grabbing cells in immune organs, liver or even brainstem got the message. But then maybe you wouldn't expect any very fancy pattern of miRNAs?

In ME/CFS we do not so far have any indication that any very unusual cells, like endometrial cells, are signalling. Which makes me still sceptical that they would ever be an miRNA signature. On the other hand maybe miRNA analysis might at least tell us about immune cell populations that at present we cannot track by blood tests but which are behaving badly in a way that is reflected in miRNA output. My memory is that there have been studies from Canada but I haven't heard more about that recently.