Up until the time she gets her first period to menopause, the average woman will go through 450 periods in her lifetime, which range between 2–7 days, although most lean towards the latter end. On average, that adds up to nearly a decade of her life that a woman is menstruating.
For many women, this means a decade of suffering. A decade of intense, unmanageable pain, bottles and bottles of medication, and an inability to function and engage in normal activities.
One of the contributors to this pain is endometriosis. A condition that sounds like a foreign word to many individuals yet affects nearly 1 in 10 women.
Endometriosis occurs when endometrial tissue is found in other parts of the abdominal cavity, including the ovaries, fallopian tubes, or tissues lining the pelvis. In some cases, endometrial cells can even travel to the chest cavity and spread to the lungs. No matter where the cells are, they still run on the same cycle as the endometrium which sheds and bleeds out through the vaginal cavity during menstruation. The cells are triggered by the presence of estrogen, a reproductive hormone. This means that no matter where endometrial cells are, from the ovaries or the lungs, they cause pain, inflammation, internal bleeding, and the formation of nodules, cysts, and scar tissue.
Women with endometriosis can face intense pain before, during, and after menstruation, alongside fatigue, heavy bleeding, painful bowel movements. Endometriosis is estimated to cause up to 70% of all chronic pelvic pain in women. 30–50% of women with endometriosis are even infertile.
Despite the impact it has, the average woman in the United States will suffer from endometriosis for up to 10 years before receiving a diagnosis.
In order to receive an official diagnosis for endometriosis, women have to undergo invasive procedures. Current diagnosis procedures include pelvic examinations, invasive vaginal ultrasounds, a hysteroscopy, or a laparoscopy — a surgical procedure in which a doctor makes incisions on the abdomen and inserts a camera into the abdominal organs. These procedures are costly, risky, and immensely uncomfortable for women, many of whom already have intense pain and discomfort due to their condition.
Many researchers, scientists, and even the World Endometriosis Association have established that the development of accurate non-invasive tests is one of the primary research priorities of endometriosis. Although research involving endometriosis biomarkers is still being conducted, many biomarkers have shown varying levels of potential in endometriosis detection. In developing a biosensor to diagnose endometriosis, we can have a profound impact on improving patients’ life quality and reducing healthcare and individual costs.
Current Biomarkers For The Detection Of Endometriosis
There are dozens of biomarkers that have been considered in the detection of endometriosis, whether in peripheral blood, urine, or blood, and range from cytokines including TNF-a, tumor necrosis factor-alpha, to vitamin D binding protein. When many plausible endometriosis biomarkers have been investigated for decades, but some common, yet not fully studied, endometriosis biomarkers are cancer antigen-125, cancer antigen-199, interleukin-6, and urocortin.
CA-125 is a very common blood biomarker for endometriosis testing and has been studied extensively. In 1998, researchers published a meta-analysis on the performance of CA-125 in the detection of endometriosis, comparing the performance of serum CA-125 measurement between women with mild endometriosis (Stage I or II) and severe endometriosis (Stage III or IV). The meta-analysis concluded that researchers can conduct two analyses on CA-125 serum; one analysis can estimate the possibility of the presence of endometriosis given a certain CA-125 concentration, and the second analysis can estimate the probability of having either acute or mild endometriosis. However, research has shown that levels of CA-125 vary throughout the menstrual cycle. Despite natural fluctuations, levels of CA-125 in women with endometriosis are typically within a higher range, especially at the very beginning of the menstrual cycle. In one study, researchers measured CA125 concentrations in the serum of both normal women and endometriosis patients during menstruation and on other days in the menstrual cycle. The age range for the normal women was 18–32, and the range for women with endometriosis was 22–34. Researchers found CA125 levels in the women without endometriosis between days 2 and 4 of the menstrual cycle to be between 5.4 and 20.5 U ml^-1 and in patients with endometriosis, this range was between 22.3 and 98.4 U ml^-1. On days 10 to 15 of the menstrual cycle, the CA125 concentrations in the serum of women without endometriosis was between 5.5 and 24.2 U ml^-1, and in women with endometriosis, it was between 8.0 and 23.5 U ml^-1. Therefore, if using CA-125 as a biomarker for endometriosis, we would not be able to diagnose patients without knowing what stage of the menstrual cycle they are in.
CA-199 is another common antigen that has been shown to be elevated in patients with endometriosis and is commonly used in the detection of pancreatic cancer. Some research shows that the cutoff thresholds of CA-199 range quite widely, from greater than 7.5 to 37.0 IU/mL, while other studies have shown that CA-199 level has a high predictive value for the severity of endometriosis, as it is localized within the epithelial cells of ovarian chocolate cysts, or endometrioma. Regardless, most studies agree that CA-199 levels are elevated in patients with Stage III and Stage IV endometriosis, if not Stage I or Stage II endometriosis. While on the whole, the sensitivity of the CA-199 tests for endometriosis is lower than the sensitivity of CA-125, there is sufficient evidence to conclude CA-199 can be a plausible and effective biomarker when testing for endometriosis, given that we are able to establish a more specific and suitable cut-off threshold, thus increasing the sensitivity of the test.
In testing for endometriosis, cytokines have a large role in the implantation of endometriotic foci, where endometrial glands and stroma grow in the pelvis or in other sites outside the endometrial cavity. Research has shown that a wide pattern of cytokines are involved during endometrial cells implantation, proliferation, and forming of endometriotic lesions, as they reduce immunologic surveillance in a given area that identifies and destroys endometrial cells. Researchers have found various sensitivity predictions for using IL-6 cytokines to detect endometriosis, leading to some inconsistencies within results. While researchers have found fairly high sensitivities in certain experimentation, others have measured it in association with other proteins and cytokines, including tumor necrosis factor alpha and serum soluble CD163. Therefore, in using IL-6 as a biomarker for endometriosis, it is more feasible to focus on the diagnostic efficacy of IL-6 combined with other cytokines or to explore cytokines such as tumor necrosis factor alpha, which shows more possibility in more accurately detecting endometriosis in comparison to mixed results from IL-6.
Urocortin is a protein belonging to the corticotropin-releasing factor family of proteins, participating in the regulation of anxiety, learning, memory, and neuroprotection. However, it has also been shown to be highly expressed in the secretory phase of the endometrial cycle, the second half of the cycle, as progesterone stimulated by the luteinizing hormone is the dominant hormone over estrogen, preparing the corpus luteum and endometrium for possible implantation of a zygote. Urocortin is expressed mainly by endometrial cells after decidualization of the endometrial stroma, where the endometrial cells react to the progestins produced by the ovary and fortify the uterine lining. While urocortin has been shown to have some relation in the pathogenesis of endometriosis, it is unclear if urocortin is a reliable biomarker as they show promise, yet data on urocortin is scarce and there is little assessment on the accuracy of their measurements in larger studies.
Circulating Endometrial Cells (CECs)
Since 2014, researchers have established that circulating endometrial cells in the peripheral blood prove to be a biomarker for endometriosis detection with high sensitivity results, which are comparatively high in relation to other biomarkers, including CA-125 glycoproteins. However, there has been minimal clinical research on circulating endometrial cells, or CEC’s as an endometriosis biomarker as it is still a relatively new concept. In addition, we are still unable to determine the exact quantities of CEC’s that can be captured by current microfluidic chips. Exploring the relationship between microfluidic technologies and the capture of CEC’s can define the possibility of CECs as a clinical endometriosis biomarker.
Scaling Electrochemical Immunosensors For Endometriosis Detection
Given the various possible endometriosis biomarkers which have been studied by researchers, alongside other biomarkers which show immense potential towards being incorporated into a biosensor for rapid endometriosis diagnostics, several specific researchers within the past 1–2 years have begun to dive deeper into the potential endometriosis biosensors hold.
An immunosensor is a type of biosensor in which a specific target antigen is detected by the formation of a stable immunocomplex between antigen and antibody, and generates a measurable signal given by a transducer.
A study published in 2020 developed a label-free electrochemical immunosensor using one-step electrochemical deposition of gold nanoparticles (AuNPs) and reduced graphene oxide (RGO) nanocomposites to detect the CA-125 antigen as an endometriosis biomarker.
In this study, the researchers used gold nanoparticles, as they have been used more frequently in biomedical nanotechnologies, due to their speed of synthesis, high conductivity, and efficiency. In addition, graphene nanocomposites were used for their excellent mechanical properties, including a large surface area, high electrical conductivity, and high biocompatibility. The functional group (the chemical group of a molecule that gives in distinct properties) of the reduced graphene oxide, allows for the interaction with metal nanoparticles and thus creates an electrochemical sensor.
The researchers found that the results from this immunosensor were very closely linked to the CA-125 concentrations as found by the ELISA, enzyme-linked immunosorbent assay, which is currently the gold standard to detect and quantify substances, including antibodies, antigens, proteins, glycoproteins, and hormones. The CA-125 concentrations increased respectively depending on the stage of the endometriosis patient (Stage I-IV), although the sensor lost a significant amount of its efficiency after being stored and reused.
Around the same timeframe, a different group of researchers developed an electrochemical immunosensor to detect a novel glycodelin biomarker. Recent research in the past several years has categorized glycodelin, a type of glycoprotein expressed during pregnancy, amniotic fluid, and in secretory endometrium, not only as a vital glycoprotein necessary for reproductive health, but also specifically expressed during malignancies, including gender-specific cancers such as endometrial cancer, ovarian cancer, and breast cancer. While it is non-specific to endometriosis, it may play a possible role in diagnosis, as studies have found that endometriosis patients have significantly higher levels of glycodelin-A concentrations, and maybe a potential biomarker for the early detection of Stage I or Stage II endometriosis.
At the same time, the same group of researchers built a very similar immunosensor around the same time; a bio-nanocomposite based highly sensitive and label-free immunosensor for endometriosis diagnostics. In this study, the researchers duplicated many of the same processes in order to develop the biosensor, first creating the immunosensor, placing it upon an electrode, using square wave voltammetry to measure interactions between the antigen and antibody, establishing optimal parameters for experimentation, and then testing the immunosensor on blood samples of women with endometriosis. The difference between the two studies was the biomarker being measured, and certain small steps that varied in measuring the appropriate biomarker. However, the process and many of the measuring techniques remained the same.
In the second study measuring CA-199 concentrations, researchers built multi-walled carbon nanotubes and magnetite nanoparticles (MWCNT-Fe3O4) and dispersed them in chitosan, a fiber extracted from insects and crustacean shells, to create a bio-nanocomposite. Multi-walled carbon nanotubes with magnetite nanoparticles prove to be the most effective in building a biosensor, as both MWCNT and chitosan composites have been used more frequently in biosensor applications due to their chemical stability, mechanical strength, and cohesive properties which link the antibodies and target analyte. However, chitosan has poor electroactive properties when interacting with MWCNTs, but several studies have shown that magnetite particles have attractive chemical and physical properties which enhance the properties of the MWCNT. They are biocompatible, have high electrocatalytic activity, but are not toxic, making them useful materials for the immobilization of biomolecules, chemically binding them to the surface of the biosensor. In particular, Fe3O4 (iron oxide) nanoparticles allow for proteins to exchange electrons directly with an electrode, which greatly improves the sensitivity of the electrochemical biosensor, which shortens the detection time of the biomolecules. Regardless, this is the first study to develop and establish a biosensor for the detection of CA-199 antigens in relation to endometriosis using a CS-MWCNT-Fe3O4 based electrochemical sensor.
Then, a film of CS-MWCNT-Fe3O4 nanocomposite was fabricated onto a glassy carbon electrode, thus immobilizing the CA-199 antibody, which would allow for the CA-199 antigens to be captured at very low levels. To clarify, the CA-199 antibodies are proteins that are produced by beta cells in the pancreas, in response to exposure to anti-CA-199, which are antigens. Each antibody contains an antigen-binding site in order to trap and eliminate antigens from the body.
The researchers examined the crystalline structure of the Fe3O4 nanoparticles, and the CD-MWCNT-Fe3O4 nanostructure through XRD analysis, or X-ray diffraction analysis, which is used to identify the crystalline phases present in a material and thus reveal information about the chemical composition. The XRD diffraction peaks reveal the position of atoms within a lattice structure.
Then, they used Fourier-transform infrared spectroscopy (FT-IR) spectroscopy, a technique used to obtain an infrared spectrum of absorption value. In this case, researchers used it to characterize the interaction among the chitosan, the MWCNT, the CS-MWCNT structure, the Fe3O4 particles, and the CS-MWCNT-Fe3O4.
In essence, the electrochemical immunosensor contains nanocomposite films of the CS-MWCNT-Fe3O4. The film is on an electrode and is used to immobilize antibodies, in this case, anti-CA-199, through glutaraldehyde, a sterilant, and a redox probe, which measures the oxidation-reduction potential.
The interaction between the CA-199 antibodies and antigens was measured using square wave voltammetry (SWV). SWV is one of the fastest and most sensitive pulse voltammetry techniques, where the differential electrical current is measured between the ends of two forward and reverse pulses. However, the current is measured between a series of pulses across a linear baseline, which minimizes the measurement of the background, or the charging, current.
In this case, the amount of CA-199 antigen reacting with the CA-199 antibodies decreased the anodic peak currents, which occurs when all of the substrate at the surface of the electrode has been oxidized, or has lost electrons. This occurs because the antigen has blocked the surface of the electrode, and thus, the electron transfer. Therefore, the concentration of CA-199 antigens can be determined based on the concentration of CA-199 antibodies and the anodic peak current of the SWV.
After the anti-CA-199 antibodies are immbolized on the CS-MWCNT-Fe3O4 film on the electrode, the redox peak current (reduction and oxidation peaks) was decreased, indicating the successful attachment of antibodies on the structure. The peak current was again decreased after the CA-199 antigen was immobilized on the electrode surface, as the CA-199 on the electrode hindered the electron transfer.
The proposed immunosensor designed in this study was optimized by several parameters, including the pH measurement, concentration of antibody, incubation temperature, in order for better electrode performance. The study found that the pH had a major impact on the biosensor as it influenced the electrochemical behavior of the film and the stability of the immobilized antibodies. The peak current slowly decreased between a pH of 6.0 to 7.4, but increased with an increase in pH between 7.4 and 8.0, showing that the anti-CA-199 antibody denatured (changed its protein structure, making it unable to bond with the CA-199 antibody) in a strongly acidic or alkaline solution, and thus, the optimal pH for a CA-199 immunosensor would be approximately 7.4. Furthermore, the most suitable temperature for the antibody-antigen interaction was found to be approximately 35 °C, as the peak current increased in higher temperatures.
Under optimal conditions (pH of 7.4, an antibody incubation time of 40 minutes, an antibody concentration of 16 μg/mL, an incubation temperature of 35 °C, and an antigen and antibody binding time of 30 minutes), the peak current decreased in correspondence with the increasing concentration of the CA-199 antigen, showing that the antigen and antibody are successfully binding on the electrode surface.
Researchers tested the bio-nanocomposite sensor on women in India who had not received any medical or hormonal treatment in three months, and women who had no prior history of any gynecological surgery including chocolate cyst, lower pelvic, or abdominal surgery. Researchers collected venous blood samples from the women as the substrate for the nanocomposite biosensor.
After collecting serum samples from endometriosis patients, 5, 10, 15, and 20 ng of recombinant CA-199 antigen was added into serum blood and measured by SWV, and the electrode was used directly for the detection of CA-199 antigen from the control and endometriosis serum. The results were compared with a standard ELISA, and researchers found that the immunosensor provided statistically significant results in the detection of CA-199 for endometriosis diagnosis.
In essence, the immunosensor designed in this study proved to be efficient, providing label-free, selective, and sensitive detection of CA-199, with a wide detection range between 0.001 to 100 ng/mL CA-199. Compared to ELISA, enzyme-linked immunoassays, this approach could be much more affordable and scalable for clinical applications, and even for point-of-care applications. This device can also be stored and reused, as the study found that the immunosensor retained 99% of the original peak current after being stored at a pH of 7.4 at 4°C for 15 days. This sensing system is selective, accurate, provides relatively rapid results, and most importantly, can be manufactured without extreme difficulty. Researchers noted that they are optimizing the possibilities to connect this immunosensor with microelectronics, in order to develop a portable CA-199 immunosensor needed for point-of-care diagnostics.
Developing A More Dependable Diagnostic Test
While research in developing reliable biosensors to detect endometriosis biomarkers is still very novel and has not been widely explored, it is an emerging field of research that shows potential to revolutionize point-of-care diagnostics.
However, there are several barriers that stop our progress in being able to scale these technologies and systems.
One of the most prominent is the lack of established biomarkers and thresholds. While many biomarkers have been studied in relation to their role in endometriosis, very few have been studied for extended amounts of time, including CA-125 and CA-199. Most biomarkers studied only show potential in endometriosis diagnostics, and need to be tested in a wider range of women and in more research to establish a correlation with endometriosis. Furthermore, many of these studied biomarkers do not have significant thresholds through which endometriosis can be diagnosed. In conducting further research with certain biomarkers, we have to be able to establish a threshold at which these hormones, proteins, cells, or other structures can accurately and reliably diagnose endometriosis, rather than simply detecting it, with the highest possible sensitivity.
In order to better establish diagnoses, we can also study how these biomarkers work in relation to each other, and potentially develop tests that look at multiple related biomarkers in order to provide more sensitive diagnoses.
At the same time, we can’t focus solely on the biomarkers themselves. In developing such biosensors to detect endometriosis, we also have to consider which biomarkers can be most easily measured by sensors, thus increasing the ease with which we can manufacture these sensors and the speed with which we can conduct these diagnoses.
After establishing significant thresholds for biomarkers, we can build more efficient, low-cost, and scalable sensors to better diagnose endometriosis. In addition, we can scale these technologies in order to monitor levels of biomarkers over longer periods of time, and integrate these systems with AI, thus providing individuals with a place to store and consistently check on their health.
By building fast, efficient, and integrated systems, we can improve the pace at which we detect, diagnose, and treat reproductive disorders.
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