Biomarkers for the noninvasive diagnosis of endometriosis

Introduction

Endometriosis (EMS) is a widely prevalent hormone-dependent disorder that affects the female reproductive system. It is defined by the growth of endometrial-like tissue (including glands and endometrial stroma) outside the uterus, typically within the peritoneal cavity⁽¹⁾, usually with an associated inflammatory process⁽²⁾. These abnormal growths can be found in various anatomical areas such as the peritoneum wall, ovaries, rectosigmoid colon, and bladder. In some cases, endometrial tissue may also be present in distant organs like the lung, liver, pleura, diaphragm, eye and the brain. Different types of endometriosis include superficial peritoneal endometriosis, ovarian endometriomas, deep infiltrating endometriosis (DIE), and extragenital endometriosis⁽³⁾. The breathtaking reality is that endometriosis impacts a staggering 176 million women across the globe, which amounts to approximately 6-10% of women in their prime childbearing years⁽⁴⁾. The main symptoms of this condition include pelvic pain, which can manifest as dysmenorrhea (painful menstrual cramps), dyspareunia (pain during sexual intercourse), painful defecation or urination, and painful ovulation. Infertility can also occur due to lesions in the fallopian tubes, adhesions, or the release of toxins by the ectopic cells. Some possible complications include bowel occlusion, hydroureteronephrosis (fluid buildup in the kidneys and ureters), ovarian abscess, rectorrhagia (bleeding from the rectum), and hematuria (blood in the urine). It is important to note that the symptoms vary in intensity and may not always correlate with disease severity. In some patients, the disease can be asymptomatic⁽⁵⁾. There are various hypotheses regarding the origin of the disease, and these can be categorized into four major groups: transport, coelomic metaplasia, embryonic cell rests, and immunological theories. The coelomic metaplasia theory explains the occurrence of endometriosis in men, while immunological theories shed light on why the disease persists. Based on our current understanding, we believe that the development and persistence of the disease are influenced by multiple factors working together. It is highly probable that EMS is a condition with a complex cause, involving genetic predisposition, prenatal exposure to endocrine-disrupting chemicals, the microbiome, the immune system, and sex hormones⁽⁴⁾. The exact causes and risk factors of endometriosis are still not fully understood, but several factors have been scientifically studied⁽⁵⁾. One widely accepted theory is the retrograde menstruation, where endometrial tissue is expelled into the peritoneal cavity during menstruation⁽⁶⁾. This theory is based on the increased incidence of endometriosis in women with outflow obstruction. However, since retrograde menstruation is common in 90% of women, it is likely that there are abnormalities in the eutopic endometrium itself in EMS which predispose to the formation of ectopic deposits⁽⁷⁾. There may also be a link between endometriosis and pelvic infection, as suggested by a retrospective study⁽⁸⁾. Other chronic and autoimmune diseases have been associated with endometriosis. Having a mother who also had EMS increases the risk, although genetic predisposition is not the sole contributing factor. Certain factors, like lower birth weight, early age at menarche and shorter menstruation cycles, have also been linked to a higher risk of EMS⁽⁹⁾. The modern lifestyle, such as decreased pregnancy rates and nulliparity, contributes to the increased incidence of endometriosis⁽¹⁰⁾. Environmental toxins like polychlorinated biphenyl and dioxin, as well as alcohol and caffeine intake have been associated with a higher risk. There are other indirect risk factors of EMS, such as skin sensitivity, night shift work, and certain dietary factors, but further research is needed to establish clear associations⁽¹¹⁾.

The diagnostic process for endometriosis can be challenging, because the symptoms of the disease are nonspecific and the available diagnostic tools are limited. While techniques like ultrasound, magnetic resonance imaging, and other imaging methods can be useful for diagnosing severe and visible cases of ovarian and deep infiltrating EMS, histological confirmation is still necessary⁽¹⁾. The gold standard for confirming an EMS diagnosis is laparoscopic surgery with histologic examination after a biopsy⁽¹⁾. However, this surgical approach has drawbacks, including the risks related to the procedure and potential complications from anesthesia⁽⁴⁾. Due to the severe and debilitating consequences of endometriosis, there is a significant need for a diagnostic biomarker that is less invasive⁽⁴⁾. In the past decade, extensive research has focused on exploring novel diagnostic tools aimed at detecting this debilitating disorder in its earliest stages⁽¹²⁾.

Biomarkers

To begin, it is important to grasp the concept and the importance of biomarkers. A biomarker is a distinct characteristic that is measured to identify normal biological processes, pathological processes, or the response to exposure or intervention, such as therapeutic treatments⁽¹³⁾. A diagnostic biomarker serves as a distinctive characteristic that can be utilized to detect or confirm a disease or condition, or to identify individuals with a specific subtype of the disease. In order to make an objective decision, all available evidence regarding a biomarker candidate must be thoroughly evaluated, including the potential benefits and risks of its use⁽¹⁴⁾. The selection of an appropriate control group holds significant importance when choosing biomarker candidates. Since endometriosis is a disease that exhibits a wide range of appearances, it is often misdiagnosed as various other inflammatory gynecological or urological conditions. Therefore, it is crucial to select a diverse control group that fulfills the criteria for the highly precise diagnosis of endometriosis⁽¹⁴⁾. Recent Cochrane studies have concluded that no noninvasive biomarker candidates currently exist that can replace the invasive laparoscopic surgery in clinical practice⁽¹⁵⁾. In this report, we outline a selection of highly promising biomarker candidates that can be used to diagnose conditions with minimal or noninvasive methods. We also assess their potential to serve as a viable alternative to laparoscopy diagnosis⁽⁴⁾.

Cancer antigen (CA)-125 is a membrane glycoprotein present in the female reproductive tract that plays a role in preventing the attachment of infectious agents to cells. It is an FDA-approved factor used in assessing the risk and monitoring the treatment of ovarian cancer. In women with endometriosis, CA-125 levels are significantly higher compared to healthy women. However, CA-125 alone is not a reliable diagnostic marker for endometriosis. While it can indicate inflammation and changes in the female genital tract, its presence throughout the body makes it less specific for EMS. Other markers, such as CA 19-9, CA 72-4 and human epididymis protein 4, have been tested individually or in combination, but none are sensitive enough for the early detection of EMS. Combining CA-125 with other markers may improve accuracy, but this approach may be costly or impractical in clinical settings. During the secretory phase, endometrial glands produce a protein called glycodelin-A, which has multiple functions, including immune suppression, regulation of blood vessel growth, and programmed cell death. The levels of glycodelin-A increase in the blood of EMS patients. While it is not a suitable biomarker on its own, combining it with other factors has the potential to create an effective biomarker panel. However, neither CA-125, nor glycodelin-A alone or in combination are sensitive enough to replace surgery as the gold standard for diagnosing EMS^(4,16), but it is still the most recommended biomarker for the diagnosis and monitoring of endometriosis⁽¹⁶⁾.

Angiogenesis is a vital factor in the progression of ectopic lesions in endometriosis. The most exten­sively stu­died proangiogenic factor in EMS is vascular endothelial growth factor (VEGF). VEGF is widely recognized as a major driver of angiogenesis and permeability in this disease. During menstruation, EMS patients showed significant overexpression of VEGF A, VEGF 121 and VEGF 189 factors compared to the control group. This finding could help explain the disease’s mechanism and could potentially serve as biomarkers, but further research is needed^(15,16).

Pigment epithelium-derived factor (PEDF) is a glycoprotein with various biological functions, including antiangiogenic, neuroprotective, anti-inflammatory and immunosuppressive properties. Previous studies found decreased levels of PEDF in the peripheral blood of EMS patients⁽¹⁷⁾, but more research is required to determine if PEDF can be a suitable biomarker. While there are encouraging findings on angiogenic factors, more data are necessary to measure their specificity and effectiveness in endometriosis. Additionally, studies should include patients with gynecological tumor diseases, since these angiogenic factors are also significant in tumor development. A sensitive biomarker candidate must be able to differentiate between endometriosis and other gynecological tumors^(4,16).

Superoxide dismutase and glutathione peroxidase blood levels (oxidative stress markers) were measured using assay kits, but their combined sensitivity was only 78%, making them unsuitable for clinical use⁽¹⁸⁾. However, the marker soluble tumor necrosis factor alpha receptor (sTNFR-I) showed promise in detecting early-stage EMS, with 75% specificity, which is noteworthy as most markers can only detect advanced stages of endometriosis^(4,16).

Immunomodulatory factors, like galectin-1, galectin-3 and galectin-9, along with inflammatory factors, such as tumor necrosis factor alpha, interleukin-1 beta, interleukin-6, interferon gamma, soluble ICAM-1 and high-sensitivity C-reactive protein (hsCRP), are not sufficiently specific to diagnose the early phase of EMS. However, ample evidence suggests their involvement in the pathomechanism of endometriosis⁽¹⁶⁾. The same was observed with autoimmune markers: anti-tropomodulin (TMOD) 3b, anti-TMOD3c, anti- TMOD3d, anti-tropomyosin (TPM)3a, anti-TPM3c and anti-TPM3d⁽¹⁹⁾, and hormonal biomarkers: urocortin (UCN), the combination of activin A and follistatin, even though the latter have proven to be highly sensitive diagnostic candidates for ovarian endometrioma⁽¹⁵⁾. Another opportunity is the screening for EMS-associated mitochondrial DNA (mtDNA). Mutation frequency in mtDNA is high, and the repair capacity is limited, making mtDNA an excellent source of biomarker candidates⁽⁴⁾.

One of the most promising areas of EMS diagnosis is represented by genomics and epigenomics. In one study, women with endometriosis had significantly downregulated microRNA levels of miR-17-5p, miR-20a, miR-22 and miR-199 compared to the control group⁽²⁰⁾. The miRNA let-7 is reportedly involved in abnormal endometrial growth and EMS⁽¹²⁾. Also, let-7d can be a reliable candidate with high accuracy, according to a study. It should be mentioned that some markers – MiR-200a, miR-200b and miR-141 – are dependent on the circadian rhythm, being lower in the morning⁽²⁰⁾ and on the menstrual cycle⁽²¹⁾. Taking the blood sampling time into account is crucial when studying circulating miRNAs as biomarkers, as indicated by the latest studies⁽²⁰⁾. Most studies recommend collecting samples in the afternoon or evening for a more sensitive and correct result⁽²⁰⁾. Also, recent studies report that these markers can be found not only in plasma but also in other biological fluids such as the peritoneal fluid, saliva and vaginal fluid; the last two could be a candidate noninvasive solution to overcome the current barriers to the diagnosis of endometriosis⁽¹²⁾. By the way, the circulating levels of miRNA and RNA in the samples proposed before can show different values in the same patient, this fact having a logical explanation. For example, the molecular composition disparities between serum and plasma have been extensively documented. Wang et al.⁽²¹⁾ observed distinct variations in RNA levels when comparing the miRNA profiles of serum and plasma. They determined that certain miRNAs and other RNAs are released during coagulation, potentially affecting the accurate assessment of circulating miRNAs. Therefore, the study suggests that plasma should be the preferred sample for investigating circulating miRNAs in order to minimize the interference caused by coagulation-related RNA release⁽²²⁾. The detection of differential expression in six miRNAs in endometriosis patients peritoneal fluid, compared to women without the condition, is noteworthy. This suggests that specific miRNAs present in the peritoneal fluid may have a role in the development of endometriosis. However, the invasive nature of collecting this fluid through surgical means limits its practical use⁽²²⁾. That’s why it was necessary to look for more accessible samples. 

A noteworthy development has emerged from a recent ENDO-miRNA study (2022), which introduced a groundbreaking saliva-based diagnostic miRNA signature for endometriosis. This remarkable achievement characterizes a significant milestone, as it is the first of its kind. Utilizing this noninvasive tool may significantly improve the early detection of endometriosis, thereby benefiting individuals with greater accessibility within any healthcare system⁽¹²⁾.

Unlike traditional methods that depend on blood samples, which often involve complex logistics⁽²³⁾, this saliva-based approach is cost-effective and easily scalable to accommodate varying levels of demand. Moreover, its collection can be conducted at any location by anyone. Saliva proves to be an ideal bodily fluid to extract disease biomarkers due to the robust stability of miRNAs it possesses, unaffected by coagulation⁽²²⁾. In addition, saliva consistently maintains sufficient RNA content compared to other bodily fluids^(12,22,23).

The study successfully established a diagnostic signature for endometriosis by meticulously analyzing 200 saliva samples, showcasing impressive accuracy, sensitivity and specificity⁽¹²⁾. Such results compel us to reassess the current diagnostic strategy which entails laparoscopy for patients exhibiting symptoms of endometriosis. The miRNAome of these samples underwent thorough evaluation, revealing the presence of detectable miRNAs and stable reads^(22,23). Among the 109 selected miRNAs, some have already been associated with endometriosis, while others warrant fur­ther investigation. Remarkably, the remaining miRNAs are involved in various signaling pathways, thereby presenting potential therapeutic implications⁽¹²⁾.

It is important to acknowledge certain limitations within the study, including the potential existence of undiscovered miRNAs, potential biases associated with hormonal treatment, and the inclusion of patients with­out laparoscopic control. Consequently, further external validation remains crucial, particularly within adolescent populations⁽¹²⁾.

However, it is important to note that, despite these limitations, the study holds great promise as it introduces a remarkable saliva-based diagnostic tool for endometriosis. This development opens up new possibilities for enhanced diagnostic accuracy, improved accessibility, and potentially transformative therapeutic approaches⁽¹²⁾.

However, it should be pointed out that more research is necessary to validate the diagnostic value of circulating miRNAs in a large cohort of samples that consist of different types of diagnostics such as malignancy, benign ovarian cysts, and chronic inflammatory disease⁽²¹⁾.

Conclusions

Efforts to find potential EMS biomarkers have explored various fields, such as genomics, transcriptomics, proteomics, and metabolomics. However, none of the biomarker candidates discovered so far have proven to be as sensitive as laparoscopic diagnosis. Nevertheless, the extensive potential of glycomic research is supported by the high sensitivity of analytical chemistry platforms. Blood and urine tests for endometriosis biomarkers offer exceptional clinical diagnostic potential. Furthermore, these samples can be collected with minimal or noninvasive methods, providing ample opportunities for the identification of new biomarkers⁽⁴⁾.  

Corresponding author: Florica Şandru, e-mail: florysandru@yahoo.com

CONFLICT OF INTEREST: none declared.

FINANCIAL SUPPORT: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.