Advances in approaches to diagnose endometriosis

Endometriosis is a common, underdiagnosed, systemic inflammatory, and endocrine pain disorder that impacts the quality of life of millions of persons with a uterus globally. It is associated with chronic pelvic pain and an increased risk of subfertility. It has also been associated with cardiovascular disease, depression, thyroid and autoimmune disorders, ovarian and breast cancers, and poor pregnancy outcomes. Treatment of endometriosis-related pain and infertility can be medical and/or surgical, although misdiagnosis due to symptoms that overlap with other conditions and reliance on definitive diagnosis by histologic confirmation of endometriosis at surgery have contributed to a prolonged diagnosis-to-treatment timeline. A paradigm shift in endometriosis diagnosis is underway, with greater reliance on clinical and family history, physical examination, and imaging, without surgical confirmation, and then proceeding with medical and/or surgical therapies, although any one diagnostic approach alone is insufficient. Although there are no validated, specific molecular biomarkers for endometriosis, recent candidates have promise for diagnosing the disease and facilitating symptom management. This review presents current and evolving approaches to diagnose endometriosis with the goal of expediting the diagnosis-to-treatment timeframe, so patients can have confidence in a diagnosis for their symptoms, expectations set about disease management across the lifespan, and a patient-centered treatment plan promptly initiated. To provide context, the review begins with a summary of disease characteristics, followed by genetic and environmental risks for developing endometriosis, the central role of estrogen and inflammation in the pathophysiology of pain and infertility relevant to the disease and biomarker discovery, and nonsurgical approaches that are increasingly being acknowledged by professional organizations across the globe to be of value in the diagnosis of this enigmatic disease with diverse manifestations.


Introduction
Endometriosis is a chronic, complex estrogen-driven disorder where endometrial-like tissue in extra-uterine sites elicits local and systemic inflammation, fibrosis, and pain [1,2] .It is an ancient disease with symptoms of pelvic pain described through written texts and art, dating to antiquity, and treated with mostly herbs and other medicinal therapies [3] .Endometriosis affects 6%-10% of premenopausal women and teens, 60% of those with chronic pelvic pain, 80% of patients with dysmenorrhea, and 30%-50% of women with infertility [2] .Disease prevalence is likely underestimated due to a lack of patient and health care provider awareness, normalization of dysmenorrhea symptoms, especially in teens, cultural mores around menstruation and pain in women, and symptoms that are not specific to the disease and its many presentations [1,2] .Misdiagnosis rates are high (65%) due to nonspecific symptoms, and in a study of > 7000 women with the disease, 46% saw more than 5 physicians to finally get correctly diagnosed surgically [4] .These factors together contribute to the protracted time of 8-11 years from symptom onset to diagnosis and surgical treatment [4] .Before diagnosis, empiric medical therapies with contraceptive steroids and nonsteroidal antiinflammatory drugs are effective but are discontinued in 25%-45% due to escape of efficacy or intolerable side effects over time [1,2] .Endometriosis takes an enormous toll on the quality of life of those affected and disrupts educational and career aspirations, work, and personal relationships [4,5] .Little recognized is its economic impact including patient out-of-pocket expenses for diagnosis and treatment, time away from work, and costs to the health system, which in 2012 were $78B in the United States alone, tantamount to other chronic diseases [4] and differs among countries.

Characteristics of endometriosis
Endometriosis is commonly found in the pelvic peritoneum and ovary, and deeply infiltrates into pelvic organs, and infrequently in the lung pleura, pericardium, and brain [6] .Peritoneal disease mainly derives from menstrual tissue fragments refluxed into the pelvis that attach to the peritoneum, stimulate neuronal and blood vessel growth, and promote an intense inflammatory response and pain [6][7][8] (Fig. 1).Metalloproteinases, angiogenic factors, and immunologic feature are key players in disease establishment and progression [6][7][8] .While nearly all women have retrograde menstruation, a subset develops endometriosis due, in part, to dysfunctional peritoneal macrophages that cannot efficiently clear menstrual debris.Other pelvic sites derive from shed cells invaginating into the ovarian cortex or surface epithelium transforming to endometriosis-lined cysts (endometriomas) or coelomic metaplasia or Mullerian rest transformation during fetal development or postpubertally resulting in deep infiltrating endometriosis (DIE; > 5 mm subperitoneal).Remote disease derives from lymphatic or hematogenous transport, transformed bone marrow stem cells, or coelomic metaplasia [6] .
Notably, endometriosis almost exclusively occurs after puberty, stimulated by circulating estradiol (E 2 ) levels and bleeding with progesterone (P 4 )-withdrawal, as in the eutopic endometrial cycle [6] .Resident nociceptors are stimulated by the inflammatory milieu and transmit pain signals centrally [7,8] (Fig. 1).The inflammatory environment in the peritoneal fluid, lesions, and endometrium promotes altered steroid hormone receptor expression and signaling [2] .This results in variable responses to ovarian-derived sex steroids resulting in endometrial dysfunction in women with endometriosis and variable and unpredictable responses to contraceptive steroid hormones commonly used to treat the disease [1] .Recently, endometrial biomarkers for inflammation have been reported, as relevant to endometriosisrelated infertility and therapeutic interventions.

Risk of developing endometriosis and associated disorders
Genetics and the environment are believed to contribute to disease risk although there is no one gene for endometriosis and environmental underpinnings are uncertain [2] (Fig. 2).With regard to the latter prolonged exposures to estrogens [eg, early puberty, low parity, in utero exposure to diethylstilbestrol, and associations in adulthood with endocrine disrupting chemicals], as well as possible bacterial and viral infectious agents have been considered [2] .Although no specific gene mutation has been identified, there is about a 6-fold increased risk if a first-degree relative is affected, and twin studies show ~50% concordance [2] .A recent genome-wide association genotyping meta-analysis [9] demonstrated that genomic regions in women with versus without disease involve sex steroid and hormone signaling pathways and angiogenesis, greater effect sizes in more advanced disease, overlap of the estrogen receptor locus with breast cancer, as well as comorbidities with other pain and inflammatory disorders.Women with endometriosis, versus those without disease, have a higher risk of fatigue, depression, and comorbidities associated Figure 1.Schematic of retrograde menstruation and disease establishment and inflammation in the pelvis and endometrium and pain signaling from nociceptors to central targets (used with permission from Giudice [1] and Basbaum et al [7] ).Abbreviations: MCP-1, monocyte chemotactic protein-1; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitors of metalloproteinases; VEGF, vascular endothelial growth factor; TNFa, tumor necrosis factor alpha; PGE2, prostaglandin E2; NGF, nerve growth factor.
Giudice.Global Reproductive Health (2024) 9:e0074 Global Reproductive Health with inflammation and estrogen, including autoimmune and atopic disorders, thyroid and cardiovascular disease, ovarian and breast cancers, and poor pregnancy outcomes (eg, higher miscarriage, preterm birth, and pre-eclampsia) after spontaneous conception or fertility therapies [10,11] .The centrality of steroid hormones, inflammation, and genetic and environmental components are striking, and although there is much known about their roles in endometriosis pathogenesis, pathophysiology, and association with other disorders, much remains to be understood.This is particularly relevant to the search for molecular biomarker discovery for disease diagnosis.

Surgery
The gold standard to diagnose endometriosis has historically been visualization and histologic confirmation of endometrial glands, and/or hemosiderin-laden macrophages in suspected lesions biopsied at the time of surgery [6] .About two-thirds of suspected lesions are confirmed histologically [12] , although confirmation depends on the site of sampling, lesion appearance, disease stage, and variability of surgeon assessment of staging and pathologist interpretation of histology [13] .The revised American Society for Reproductive Medicine (rASRM) surgical staging [14] scores disease volume and adhesions but does not account for DIE or correlate with pain measures or predict response to medical therapies for pain or infertility.Several professional organizations are attempting to optimize disease classification.For example, the World Endometriosis Society 2017 consensus statement [15] recommends using a "classification toolbox" that includes the rASRM system and the Enzian system for deep disease and improving classification not based on surgery.It also promotes including the extensively validated Endometriosis Fertility Index which has high sensitivity and specificity for fertility outcomes after surgical treatment of disease and adhesions and has been extensively validated across the globe [16,17] .

Multiple players and a paradigm shift
Detailed patient medical, menstrual, pregnancy, surgical, family, and exposure histories, along with physical examination and imaging comprise key initial approaches to endorse a suspicion of endometriosis in the differential diagnosis of pelvic pain and infertility.More recently, the paradigm has shifted to clinical diagnosis with adjunctive imaging techniques and away from diagnostic laparoscopy before initiating medical therapies for pain and infertility.This is endorsed by several global professional organizations, including the American College of Obstetricians and Gynecologists, World Endometriosis Society, the Society for Obstetrics and Gynecology of Canada, and the European Society for Human Reproduction Embryology [18][19][20] .
Likely, others will follow suit.

Gynecologic history
Pelvic pain alone is insufficient to diagnose endometriosis, as it occurs in other gynecologic disorders (eg, primary dysmenorrhea, fibroids, adenomyosis, ovarian cysts, pelvic infection, pelvic congestion syndrome, and pelvic floor dysfunction) and nongynecologic disorders (irritable bowel syndrome, celiac disease, interstitial cystitis, musculoskeletal disorders, pelvic adhesions, and fibromyalgia), some of which coexist with endometriosis [2,8,10,19] .The most common pain symptom in women with endometriosis is chronic, cyclic pelvic pain (dysmenorrhea; Table 1), which usually worsens over time and may progress to include noncyclic pelvic pain that is unpredictable in onset, Zondervan et al [2] ).
duration, quality, and severity.Associated symptoms, along with relevant patient history and findings on physical examination, increase the index of suspicion for endometriosis.Notably, the odds ratio for endometriosis disease, based on pain symptoms alone, increases from 5.0 to 84.7 for 1 and > 7 symptoms, respectively [21] , underscoring the value of the clinical history.A family history of the disease can facilitate diagnosis in the proband, and environmental endocrine disrupter exposure history, as in pregnant patients [22] , may promote well-being and a healthy lifestyle, although it has not been validated in women with endometriosis.Although most women with endometriosis have regular cycles without abnormal bleeding, a minority have premenstrual spotting, irregular menses, and heavy menstrual bleeding [19] .These are all important components of historytaking in symptomatic women and can validate the heterogeneity of symptoms experienced by women with the disease, as well as promote investigation into other disorders contributing to these symptoms [19][20][21][22][23] .
A comprehensive clinical assessment of patient symptoms that offers pain maps and extensive gynecologic and health history intake is captured in the International Pelvic Pain Society's Pelvic Pain Assessment form [24] .While not designed to diagnose endometriosis, it can be empowering for patients to document their pain history, location, and quality, and for health care providers to review before the patient's initial visit.The Visual Analog Scale is a validated acute and chronic pain instrument with paper, laptop computer, and mobile phone-based platforms, that can facilitate data collection and entry into the electronic medical record for initial presentation and symptom course over time [25] .

Infertility
A complete workup of patient age, duration of infertility, prior pregnancies and their outcomes, associated disorders in the female partner, and male evaluation are essential [26] .Women with ovarian endometriomas may have decreased ovarian reserve, and prompt diagnosis is key in planning fertility therapies and/or surgical extirpation, if warranted.While 30%-50% of women with endometriosis have infertility, the latest Society for Assisted Reproductive Technology Clinical Online Reporting System database notes a diagnosis frequency of endometriosis of 3% [27] , a major underestimation reflecting discontinued diagnostic laparoscopies (and thus disease diagnosis) as part of infertility evaluation.Thus, a high index of suspicion is warranted for endometriosis in infertility evaluation and care.

Pelvic examination
Pelvic examination may identify endometriosis with high accuracy, although it is highly dependent on disease location, does not detect superficial peritoneal lesions, and may be unacceptable in nonsexually active women [19] .Ruling out other causes of pelvic pain and infertility is essential, as therapies for these likely differ and may require referral for specialty care.

Imaging
Imaging technologies, which are rapidly developing, are increasingly being used to diagnose endometriosis, adjunctively with patient symptoms and history [28] .Transvaginal ultrasound (TVUS) is the first-line imaging approach with high sensitivity and specificity for ovarian endometriomas, and ultrasound (US) signs can reveal pelvic structure immobility and scarring.However, TVUS performs poorly in detecting peritoneal implants.More recently, TVUS has been shown to have high accuracy, comparable to magnetic resonance imaging, for detecting DIE, with experienced sonographers, and is valuable in preoperative assessment and surgical referral [28] .A recent Cochrane meta-analysis found that TVUS has sensitivity and specificity-similar to surgical diagnosis, depending on the type of disease [18] .A model that combines patient history, symptoms, and US findings predicts with good accuracy rASRM advanced stage disease, but not minimal/mild disease [29] and thus has not been widely adopted.A recent systematic review and meta-analysis of 30 studies involving 4565 patients found that TVUS, transrectal US, and magnetic resonance imaging have comparable accuracy compared with each other and higher accuracy than physical examination alone to diagnose endometriosis [30] (Table 2).It is anticipated that algorithms integrating large clinical and imaging data sets, subsequently validated, will be useful in clinical practice (office setting and preoperatively) in the nottoo-distant future.This may also facilitate clinical trial enrollment for endometriosis-related pain in phase III trials, where surgical diagnosis currently is an inclusion criterion required by the U.S. Food and Drug Administration.

Molecular biomarkers of disease
CA 125, expressed in endometrium and endometriosis lesions, is elevated in the serum of some women with the disease [31] .While serum CA 125 > 35 units/mL has high specificity for all rASRM stages (I-IV) and specifically stages III/IV (90%, 89%, respectively), it has low sensitivity (28%, 47%, respectively) [31] , and thus has minimal clinical value.Despite extensive research over the past 2 decades and no disease-specific biomarkers identified and validated, recently, candidates have emerged that show promise; plasma and salivary microRNAs and endometrial B-cell lymphoma 6 (BCL6) [32][33][34][35] .Diagnostic classifiers developed using plasma miRNA signatures had high accuracy (area under the curve = 0.94), were validated in independent data sets, and distinguished rASRM stage I/II and III/IV from controls but not stage I/II from III/IV [32] .Of those identified with disease, 90% had pelvic pain and 10% had infertility, and notably, the diagnosis was independent of cycle phase or hormonal medications.In addition, a suite of 109 salivary microRNAs has recently been shown to diagnose endometriosis with high sensitivity, specificity, and area under the curve (96.7%, 100%, 98.3%) [33] .
Endometrium in women with endometriosis has a proinflammatory environment manifested by heightened estrogen receptor beta signaling and progesterone (P 4 ) resistance, resulting in abnormal expression of several genes and proteins in the implantation window [6] .BCL6 is abnormally upregulated in the endometrium of women with disease (and unexplained infertility some were later found to have endometriosis) [34] .In women with abnormal BCL6 protein expression, suppression of disease and associated inflammation (using gonadotropin releasing hormone analogs or progestins), or surgical ablation reportedly improves live birth rates (50% treated and 7% untreated) in women undergoing assisted reproduction in vitro fertilization-embryo transfer [35] .The utility of endometrial BCL6 expression as a diagnostic tool remains to be determined in clinical trials.
Thus, these new diagnostic candidates, based partly on endometriosis pathophysiology, show great promise and await multicenter randomized control trials for further validation and broader applications, for example, assessing disease and symptom recurrence across the lifespan.

Conclusions
It is of great importance that health care providerspediatricians, gynecologists, primary care physicians, and school nurses-recognize that endometriosis is a chronic disease with heterogeneous symptomatology and disease subtypes and unpredictable short and long-term responses and side effects of medical and surgical therapies for pain and infertility.Thus, for symptomatic patients, thorough history-taking and imaging coupled with non-invasive molecular diagnostics could set the foundation for multidisciplinary, patient-centered care [1] early in Adapted from references in review [30] .Adaptations are themselves works protected by copyright.So in order to publish this adaptation, authorization must be obtained both from the owner of the copyright in the original work and from the owner of copyright in the translation or adaptation.a Pooled performance percentages from systematic reviews/meta-analyses.AUC indicates area under the curve; LR, likelihood ratio; MRI, magnetic resonance imaging; PE, physical examination; TRUS, transrectal ultrasound; TVUS, transvaginal ultrasound.
the disease course and during its many manifestations across the lifespan.In addition to the prompt diagnosis being key to expedited initiation of therapies for well-being, it is also key to promote patient autonomy and symptom validation.Importantly, the World Health Organization has recently provided a fact sheet on endometriosis to increase awareness and foster earlier diagnosis and treatments for women and girls across the globe [36] .

Figure 2 .
Figure 2. Schematic of endometriosis risk factors and manifestations across the life course that can facilitate history-taking (used with permission from Zondervan et al[2] ).

3 .
Perform physical examination • Nodules in cul de sac • Retroverted uterus • Mass consistent with endometriosis • External endometrioma (eg, umbilical and other) • Pelvic floor spasms • Severe allodynia along pelvic floor/vulva or elsewhere • Masses not consistent with endometriosis (eg, fibroids) 4. Perform/order imaging • Endometrioma on US • Presence of soft markers (eg, sliding sign) • Nodules and masses • Adenomyosis and fibroids (although these may be present with endometriosis) • Malignancies Used with permission from Agarwal et al.Clinical diagnosis of endometriosis: a call to action.Amer J Obstet Gynecol 2019;April:354-364.Copyright 2019 The Author(s).Published by Elsevier Inc.This is an open-access article under the CC BY-NC-ND license.US indicates ultrasound; EDC, endocrine disrupting chemicals.

Table 1
Algorithm for clinical diagnosis of endometriosis.

Table 2
Performance of various imaging modalities and PE in the diagnosis of endometriosis.