Menstrual Blood Biomarkers for the Noninvasive Diagnosis of Endometriosis: A Review

doi:10.21203/rs.3.rs-4923540/v1

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Systematic Review

Menstrual Blood Biomarkers for the Noninvasive Diagnosis of Endometriosis: A Review

https://doi.org/10.21203/rs.3.rs-4923540/v1

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Diagnosis of endometriosis remains challenging, as the diagnostic delay is still encountered. This phenomenon is in accordance with the reported cases, in which the definitive diagnosis is approximately issued by 7—12 years delayed onset of symptoms. Many findings have reported several putative biomarkers from various resources, but no single biomarker displayed enough accuracy to be used outside the research setting. As the primary source of retrograde menstruation, identifying the constituents of menstrual blood may provide critical information on the pathogenicity and dysregulation occurring in the endometrium milieu. Indeed, the recent findings on menstrual biomarkers have demonstrated significant progress in the quest for new alternative sources to diagnose endometriosis. Further research with a combination of different approaches and integrated panel biomarkers is needed to bring the menstrual blood biomarkers step forward to clinical application. Here, we reviewed the menstrual biomarkers that have been reported to date, unlocking the potential utility of menstrual blood to develop an early, noninvasive diagnosis of endometriosis.

Obstetrics & Gynecology

Biomarker

endometriosis

diagnosis

menstrual blood

noninvasive

Endometriosis is acknowledged as a systemic disease characterized by the presence of endometrium-like tissue outside of the uterine cavity [1, 2]. This condition is mainly associated with chronic pelvic pain and infertility, affecting about 190 million women of reproductive age worldwide [1, 3]. The prevalence remains unclear due to the limitation of definitive diagnosis, but approximately ranging from 2 to 11% among asymptomatic women, 5 to 50% among infertile women, and 5 to 21% among women with pelvic pain [1]. In addition to causing a significant economic burden, endometriosis also impairs poor mental health and is correlated with decreased quality of life [4, 5].

Diagnosis of endometriosis remains challenging, as the diagnostic delay is still encountered [6, 7]. This phenomenon is in accordance with the reported cases, in which the definitive diagnosis is approximately issued by 7—12 years delayed onset of symptoms [7, 8]. Several factors are considered to exacerbate this delay, including symptom normalization, uncertainty of pathogenicity, and a lack of clinical guidelines [6, 9, 10]. The heterogeneity and the confounding of potential comorbidities related to other gynecological and non-gynecological symptoms also complicate disease recognition [10]. In addition, the absence of a valid and reliable noninvasive diagnostic test presumably affects the diagnostic delay [9]. The consequences of this delay may cause extended periods of pain, as well as foster disease progression that compromises the life impact [6, 8].

The urge for the noninvasive diagnosis gave rise to several techniques, suggesting a shift from the surgery-based method. Recently, biomarker discovery has been taken into account to hasten the early diagnosis of endometriosis. The inevitable, long, complex stages are involved, from initial biomarker discovery to a clinically approved biomarker assay [11]. Many findings have reported several putative biomarkers from various resources, such as peripheral blood, urine, endometrium, saliva, and peritoneal fluid –determined by their accessibility and molecular features. However, numerous systematic reviews indicated that no single biomarker of those resources displayed enough accuracy to be used clinically outside the research setting [12–15].

Menstrual blood is a relatively unexplored area for diagnostics, although it conceives significant potential as a source of biomarkers [16, 17]. Menstrual blood provides a noninvasive, readily accessible source of biological material and covers a broad sphere for investigating common gynecological conditions [16, 18, 19]. Proteomics studies on menstrual blood have revealed several biomarkers for extensive disorders, including endometriosis, cervical cancer, failed implantation, and recurrent miscarriage [19, 20]. These data support the potential utility of menstrual blood-based testing as an alternative for diagnostic or therapeutic analysis. Furthermore, such an approach could enhance opportunities for early disease detection and regular health monitoring [20].

Several studies have revealed the distinctive profile of menstrual blood using the 'omics' approach. A proteomic study by Yang et al. reported 385 proteins that are unique to menstrual blood compared to peripheral blood and vaginal fluid [21]. Similarly, Wyatt et al. also found eight elevated proteins exclusively present in menstrual blood [16]. The cytokine profiling by Guterstam and Naseri further elaborated on a higher concentration of cytokine detected in menstrual effluent [18, 22]. Moreover, several studies also revealed the unique characteristics and compositions of stem cells, immune cells, organoids, and miRNA isolated from menstrual samples that strongly resemble biopsy-derived endometrial cells [19, 23–25]. Consequently, these results promote significant implications for translational studies of menstrual blood by identifying its variety of cellular and protein characteristics, unlocking the potential utility for identifying endometrial diseases [16].

According to retrograde menstruation, menstrual effluent is considered to play a role in ectopic lesion formation [26]. Koks et al. reported that tissue fragments isolated from shed menstrual effluent were preferentially adhered to the peritoneum [27]. Moreover, menstrual blood from women with endometriosis also showed a significantly higher prevalence of basalis fragments compared to healthy controls [28]. In addition, several reports also highlighted the increased proportions of endometrial stem/progenitor cells in the menstrual blood of endometriosis subjects [3, 16]. Therefore, menstrual blood biomarkers can be developed as an alternative diagnostic method for endometriosis [26].

Biomarkers of endometriosis have been extensively reviewed from several sources, such as peripheral blood, peritoneal fluid, and urine. However, only limited studies analyzed the potential utility of menstrual blood. To the best of our knowledge, this is the first review focused on menstrual blood biomarkers for diagnosis of endometriosis.

Eligible studies were searched from Scopus and PUBMED Database using the keywords "Endometriosis," "Menstrual Effluent," and "Menstrual Blood." To be included, each study had to measure biomarker levels in menstrual blood samples from endometriosis patients compared to healthy women. All studies must be restricted to patients without hormonal treatment or other medications. The studies must be presented as original articles written in English and not conducted on animal models. Commentaries, letters to editors, editorials, and conference abstracts were excluded. No time restriction was applied to the searches due to the limited number of reports. Therefore, the data were collected from every relevant study reported to date.

The review was performed to find any statistically significant difference between groups, as evidenced by the statistical analysis results reported in the selected papers. Differences were expressed as means/median ± standard deviation (SD), and a P < 0.05 was considered statistically significant. The systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (Fig. 1).

Of the 156 reports generated by PUBMED, 12 studies were selected based on title and abstract. After further analysis, three reports were excluded because of insufficient information on biomarkers. A search on Scopus exhibited 773 reports with the same search criteria, which 16 studies considered relevant. We further evaluated 11 out of 16 reports based on the data provided, as five remaining studies were either focused on the mechanism of pathology of endometriosis, profiling cell subsets of menstrual blood, or using endometrial/uterine fluid rather than menstrual blood. Finally, 20 reports in total were included in this review (figure 1).

The data extracted from the studies comprises several information, such as sample collection method (time, tools, analyte); clinical aspects/characteristics of the case-control group (number, staging, age, BMI); methodology (assay and statistical analysis); and significant findings of biomarkers (concentration or fold change). Only four out of 20 studies provide complete information on each data.

Information on disease stage was found only in eleven studies, where most patients enrolled in the case group were in stage III-IV according to the Re-ASRM classification. Although biomarker studies should encompass the minimal-mild stage, only six studies involved patients with early-stage (I-II) of the disease. Furthermore, the case group from 2 studies also included patients with adenomyosis and undiagnosed cases with chronic symptoms. On the other hand, the control group comprised women either with infertility, pelvic pain, or patients with other gynecological diseases (no endometriosis), which were confirmed by laparoscopic surgery or hysterectomy. In addition, several studies also included self-reports from women with no gynecologic history or who were considered healthy by bimanual examination and ultrasonography.

Sample collection on these studies was conducted on the heavy bleeding phase of menstruation, around days 1-3 of the period. Patients independently collect the fluid with the menstrual cup or use a syringe, cannula, or gauge needle to aspirate the blood. Several studies specifically targeted specific types of stem cells or stromal cells, which need further isolation and culturing procedures, while others directly analyzed the whole menstrual effluent. According to the findings, only 12 studies provide information about the concentration or fold change of the biomarkers.

This review included six studies that analyzed other sources of biomarkers (peripheral blood or peritoneal fluid) compared with menstrual blood. We also found three studies that examined the decidualization capacity of endometriotic stromal cells induced by cAMP treatment. Lastly, one study on endometritis was considered since the disease shared similar inflammation properties with endometriosis.

This review observed various methodologies, including ELISA, RT-qPCR, mass spectroscopy, RNA sequencing, immunohistochemistry, and radioimmunoassay. In addition, the Omics approach was applied in three studies, resulting in a great profiling of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs). However, most studies did not provide detailed information on sensitivity or specificity, so the potential utility of the biomarkers cannot be clinically evaluated.

Surface Marker

Seven studies have reported several surface markers in menstrual blood that are differentially expressed in endometriotic patients. Cluster of Differentiation (CD) markers were the most evaluated, followed by CA-125 and Podoplanin (PDPN). Among CD markers studied are CD9, CD10, CD29, CD73, CD74, CD83, CD90, and CD105. Consistent results were found in the higher expression of CD10 from two studies, where CD9 expression was contradictory with each other [29,30]. One study revealed the high expression of CD29, while other studies found the lower expression of CD74 (-3 folds), CD83 (-1.9 folds), and CD105 in endometriotic patients [26,30,31]. Inversely, CD90 or CD73 showed no significant differences in cultured menstrual effluent-stromal fibroblast (ME-SFCs) derived from endometriosis patients compared to control subjects [26].

CD9 expression in endometrial stromal cells has been reported to be associated with infertility, especially with the alteration of implantation [29]. Moreover, this marker also mediates signal transduction, growth, and motility [30]. The different results of CD9 expression in those studies remain unclear, thus suggesting further evaluation. On the other hand, CD29 has been known to be attributed to malignancy and EMT (epithelial to mesenchymal transition) process. The higher expression of this marker may reflect the tumor characteristic of stromal cells, leading to the establishment and propagation of endometriosis lesions [30]. Conversely, Lower expression of CD74, CD83, and CD105 in the menstrual blood of endometriotic patients explained their role as macrophage-associated genes. CD74 and CD83 encode the receptor of macrophage migration inhibitory factor, while CD105 regulates the binding of various ligands, including TGFβ1, TGFβ3, BMP-2, and BMP-7 [26,31].

In other studies, Takahashi et al. evaluated cancer antigen 125 (CA-125) from serum and menstrual blood. CA-125 is a glycoprotein expressed by epithelial ovarian tumors and is expected to predict the severity and pathological characteristics [32]. They consistently found significant levels of CA-125 in menstrual blood in all stages of endometriosis, while serum CA-125 was only elevated in advanced stages [33,34]. These findings further proposed the evaluation of menstrual CA-125 as a reliable marker for early diagnosis of endometriosis [33–35]. Moreover, menstrual discharge allegedly represents the endometrium milieu, while serum may reflect a broader range of diseases [33]. On the other hand, the study by Nayyar et al. reported that the menstrual blood of endometriotic patients showed a significant level of podoplanin (PDPN), a surface marker of fibroblast cells that are associated with tumor cell invasiveness [36].

Decidualization Marker

Decidualization occurs when endometrial stromal fibroblasts (ESCs) undergo differentiation to secretory 'epithelioid' cells in preparation for implantation and pregnancy [51]. The reprogramming of ESCs requires cAMP and occurs under the influence of estrogen and progesterone [51]. Insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) are the most classical markers of decidualization, and they are found to be highly enriched in amniotic fluid [52,53]. Deregulation of the IGF/IGFBP-1 system is associated with pregnancy complications and some endometrial diseases [52]. Indeed, women with endometriosis have been reported to have impaired decidualization in response to cAMP and activation of the PKA pathway [53].

We found three studies evaluating the decidualization capacity of menstrual-derived ESCs (ME-SFCs) from women with endometriosis. All studies measured the IGFBP-1 levels after cAMP treatments to assess the decidualization impairments. Expectedly, three studies found consistent results in reducing IGFBP-1 levels in stromal cells of endometriosis cases compared to control [26,36,48]. According to the study, Shih et al. proposed that endometrial stromal cells with proinflammatory, profibrotic, and senescent phenotypes were the leading cause of decidualization deficiency [48]. These findings suggested that decidualization capacity can be measured directly in stromal cells derived from fresh menstrual effluent (ME), thus supporting the utility of menstrual-based decidualization assay as a potential noninvasive diagnostic for endometriosis [36,48].

On the other hand, we also evaluated other markers of decidualization reported by three studies, two of which corresponded with IGFBP-1 studies. We found two reports on ALDH1A1 expression and one on AFT3 and NR4A1 expression. These reports concluded that ALDH1A1 has significantly repressed, while AFT3 and NR4A1 were highly enriched in menstrual-derived ESCs (ME-SFCs) from women with endometriosis compared to control [26,36,49]. ALDH1A1 encodes an enzyme that converts retinol into retinoic acid, which is necessary for endometrial cell decidualization [36]. Similarly, ATF3 and NR4A1 also play a role in endometrial decidualization and the epithelial-mesenchymal transition [49]. However, evidence from primary transcriptomic signature data from an oocyte donation model found that endometrial receptivity did not differ significantly between women with and without endometriosis and across the different stages of endometriosis [54].

Marker of Inflammation

Inflammation is critical in the onset and progression of endometriosis [55]. Functional changes in macrophages, NK cells, and cytotoxic T lymphocytes/TH17 cells in women with endometriosis result in a more immunotolerant peritoneal microenvironment, allowing disease to establish following retrograde menstruation [56,57]. As the post-differentiation route generally results in apoptosis of shed endometrial cells, tissues entering the peritoneal cavity typically facilitate immune scavenging without inducing a prominent response. However, it has been demonstrated that immune surveillance is compromised in women with endometriosis, where the innate immune system cannot respond appropriately to the displaced tissue within the peritoneal cavity. Specifically, decreased NK cell cytotoxicity, overactivation of macrophages, and the local production of cytokines and chemokines further enhance ectopic endometrial tissue growth [56].

Our review on menstrual biomarkers of endometriosis found that inflammation is the most studied marker, reported by 9 out of 20 studies. Most of these studies analyzed the cytokines and chemokines (6 reports), followed by other immune markers related to inflammatory responses. Among the cytokines that have been evaluated are IL-1β, IL-6, IL-8, IL-10, IL-11, IL23A, and IL1RN. Several cytokines were highly expressed in the menstrual blood of endometriotic patients, including IL-1β, IL-6, IL-8, IL-11, and IL1RN [29,31,38,39,47,48]. However, one study also found no significant differences in IL-6 and IL-8 expression among the case-control group [47]. In addition, the study by Miller et al. and Nikoo et al. revealed the lower expression of IL23, IL-10, IL6, and FOXP3 that are vital for T-cell differentiation, suggesting the impaired regulation of the TH17 axis in women with endometriosis [30,31].

Apart from that, alterations in chemokine expression were also highlighted in two studies. Ji et al. and Miller et al. independently reported the upregulation of C−X−C-Motif Chemokine Ligand 5 (CXCL5) and downregulation of CXCL16 in menstrual blood of endometriosis patients [31,38]. CXCL5 is a member of the CXC chemokine family involved in activating neutrophil granulocytes, while CXCL16 is known to drive macrophage activation and polarization [31,38]. In addition, several immunomodulatory factors were significantly enhanced in the endometriosis group compared to the control group. Among those factors are COX-2, HMGB-1, IDO1, EGR1, ZFP36, ID1, and ID3 [29,30,45,49]. However, evaluation of tumor necrosis factor-alpha (TNF-𝛼) from three studies found no differences among patients with and without endometriosis [29,39,46,47] but highly expressed in menstrual effluent of women with chronic endometritis [39]. Therefore, the inconsistent findings of TNF-𝛼 expression suggest further evaluation, although these factors are believed to promote inflammation and angiogenesis in endometriosis [58].

Similarly, two studies reported other markers with insignificant results, such as MPO, NAG, and HIF [29,46]. Myeloperoxidase (MPO) is an enzyme restricted to the azurophil granules of neutrophils, while N-acetyl-b-D-glucosaminidase (NAG) is broadly known to detect macrophage accumulation/activation. Both NAG and MPO evaluated in infertile women with endometriosis showed no difference in menstrual blood from women with endometriosis and control but significantly higher in menstrual blood than in peripheral blood from the endometriosis group [46].

Marker of Apoptotic, Migration, and Adhesion

Only one study analyzed the dysregulation of apoptosis in endometriosis patients using menstrual-based evaluation. Apoptosis is a process in which cellular homeostasis is maintained by eliminating the excess of the uterine functional layer, regulated by various regulatory factors. Among these regulators, both anti-apoptotic (Bcl-2 and Bcl-xL) and pro-apoptotic factors (e.g., Bax and caspase-3) play a vital role in controlling the process [59]. Specifically, the ratio of Bax/Bcl-2 has been known to be associated with the apoptosis level of diseases [29]. A study by Sahraei et al. reported that the Bax/Bcl-2 ratio was significantly lower in E-MenSCs compared to NE-MenSCs, thereby confirming the decreased apoptosis in endometriosis stromal cells [29].

Regarding the migration capacity, two studies explored the epithelial-to-mesenchymal transition (EMT) in endometriosis. EMT is a process marked by loss of intercellular connection and polarity of epithelial cells while acquiring mesenchymal properties with invasive phenotype [60–62]. A bioinformatics analysis by Chet et al. highlighted EMT as the hallmark pathway of differentially expressed genes (DEGs) in endometriosis [63]. Matrix metalloproteinases (MMPs) are the most studied among EMT markers, as upregulating MMP-2 and MMP9 were associated with tissue breakdown, which further induces migration and invasion [62]. Accordingly, two studies revealed increased levels of MMP2 and MMP9 in E-MenSCs compared to NE-MenSCs [29,44]. Furthermore, these studies also found the lower expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) in E-MenSCs, explaining how MMP levels tend to be higher in endometriosis patients [44]. However, the study by Malik et al. observed that MMP2 and MMP9 activities were shown to be similar between the two groups [64].

Other migration markers that have been reported are MGP, NANOG, OCT4, and SOX2. MGP is associated with the extracellular matrix and was found to increase in the endometriosis group [48]. On the other hand, NANOG, OCT4, and SOX-2 are correlated with stemness-related genes, which are known to promote cell survival and migration. NANOG and OCT4 had significantly lower expressions, whereas SOX2 genes are upregulated in E-MenSCs compared to NE-MenSCs [29].In addition, analysis from endometrial fluid also confirmed the differences in the expression of migration-related genes, such as the 14-3-3 gene (associated with signal transduction) and moesin (associated with cytoskeletal structure) in endometriosis patients [65].

On the other hand, cell adhesion capacity has been evaluated from vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule (ICAM-1). VCAM allegedly drives the disassembly of focal adhesions and stimulates T-cell migration. Similarly, ICAM is also implicated in migration, endometrial–peritoneal adhesion, and the survival of endometrial cells by protecting them from immune response [66]. However, there were no differences in ICAM-1 and VCAM-1 concentrations in menstrual effluent from the case-control group. ICAM-1 levels were higher in menstrual effluent than peripheral blood, while VCAM-1 showed the opposite finding [47].

Marker of Cell Proliferation and Angiogenic

The development of endometriotic lesions is typically associated with angiogenesis. This process underlies the complex regulation by angiogenic growth factors and hormones, which further activate intracellular pathways [67,68]. Vascular endothelial growth factor (VEGF) has been broadly studied in the peritoneal fluid of women with endometriosis, which is consistently detected in high concentrations and appears to correlate with the stage of the disease [43]. Accordingly, two studies on VEGF analyzed from menstrual blood shared the same results, where the VEGF was expressed at a high level in E-MenSCs compared to NE-MenSCs [29,43]. However, two other studies also reported that VEGF-A and sVEGF-R1 (sFLT) were shown to be similar between the two groups [46,64]. Apart from that, a transcriptome and proteome profiling by Penariol et al. revealed several differentially expressed genes/proteins (DEGs/DEPs) in E-MenSCs, highlighting the increased of other angiogenic genes, such as TYMP, MT2A, and COL6A2 [49].

Regarding the proliferation ability, two studies evaluated Cyclin D1 and DROSHA expression, respectively. Cyclin D1 is a mitotic cyclin that plays a vital role in many types of cancer. Indeed, Sahraei et al. found a significantly elevated Cyclin D1 (5.4 folds) in E-MenSCs compared with NE-MenSCs [29]. On the other hand, Cressoni et al. reported a two-fold decrease in DROSHA expression in the endometriosis group, representing the dysregulation of miRNA biosynthesis [50]. miRNAs have modulated critical components of endometriosis's biological pathways, including cellular proliferation, invasion, and angiogenesis [69].

Hormonal Marker

Hormonal alterations may affect the ability of endometrial cells to proliferate, attach to the mesothelium, and evade immune clearance [70]. Dysregulation of progesterone receptor (PR) and estrogen receptor (ER) has been detected in endometriosis patients, representing progesterone resistance and estrogen dominance. Furthermore, the upregulation of the ER pathway and the overactivation of aromatase activity leads to highly increased local E2 production in endometriotic cells [5,71].Coherently with these findings, four studies evaluated the hormonal marker from menstrual blood reported increased levels of estradiol (E2), P450 Aromatase, and ER-α, along with the lower expression of PR- β in the endometriosis group compared to control [29,40–42].

Analysis of endometrium shed has revealed the potential to develop an early, noninvasive diagnostic method for endometriosis. However, due to the limited evidence, an in-depth exploration of menstrual blood is needed to provide results equivalent to those of the commonly used surgery-based method. Further research with a combination of different approaches and integrated panel biomarkers is needed to bring the menstrual blood biomarkers step forward to clinical application. To date, the recent findings on menstrual blood biomarkers have demonstrated significant progress in the quest for a new alternative source of biomarkers. Moreover, given the readily accessible feature, definitive menstrual blood biomarkers suggest the future prospects for prognostic and monitoring of endometriosis, as well as evaluating the recurrence and promoting individualized treatment.

Acknowledgement

There is no financial support and conflicts of interest in this study.

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Menstrual Blood Biomarkers for the Noninvasive Diagnosis of Endometriosis: A Review

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