The Next Step Towards Endometriosis Detection - A Hands On Engineering Design Project

Endometriosis is a gynecological disorder in which tissue similar to the inner lining of the uterus grows outside of the uterus, affecting the pelvic organs [1]. The misplaced tissue responds to hormonal changes in the same way as typical uterine tissue, in that it thickens, breaks down, and bleeds during the menstrual cycle. However, due to the misplacement, there is no way for this excess tissue to exit the body. This can cause cysts, scar tissue, and adhesions to develop [2]. As a result, endometriosis causes symptoms such as heavy menstrual bleeding, pain, and infertility. Although endometriosis affects approximately 1 in 10 women worldwide, few treatment and diagnosis options are available [3]. Endometriosis shares symptoms with many other conditions such as Irritable Bowel Syndrome (IBS), Polycystic Ovary Syndrome (PCOS), pelvic floor disorders, and even stress-related dysregulation, making it difficult to pinpoint endometriosis specifically. Endometriosis is difficult to diagnose early-on and often doesn’t show major symptoms until the condition has progressed extensively [4]. This results in our identification of the need for a device that is able to detect analytes indicative of changes caused by endometriosis to indicate the presence of endometriosis during earlier stages. Currently, the gold standard is a laparoscopy, which entails making a small incision in the abdomen and visualizing the reproductive organs to see if any signs of endometriosis are present [5]. Not only is this method invasive, but it is also expensive and requires a surgeon to be involved. Our process in this customer need driven process of engineering design was predicated on including the voice of the customer in the final design [6], with the term “customer” indicating all relevant stakeholders in the medical device continuum of care [7]. To address the challenges associated with current diagnostic practices, we propose a portable, non-invasive device designed for point-of-care detection of endometriosis-related biomarkers. Existing predicate devices typically focus on single biomarkers [8], such as CA-125, and often require samples to be sent to external laboratories for analysis [9]. While such devices allow our device to have a defined regulatory pathway for substantial equivalence, our proposed design in contrast emphasizes real-time, at-home testing capabilities that provide users with immediate feedback. Intended for over-the-counter use, this system serves as a data collection platform that integrates multiple biomarkers that were previously studied in isolation to improve diagnostic accuracy [10]. By analyzing biomarkers present in urine, blood, or saliva, we hypothesize that a multi-analyte approach will enhance diagnostic specificity for endometriosis, distinguishing it from other inflammatory conditions. The long-term goal is to reduce diagnostic delays and empower users to take greater autonomy over their reproductive health. By designing a compact, affordable device compatible with multiple biomarker detection methods, this system aims to bridge the gap between laboratory research and real-world diagnostic accessibility by serving as both a clinical screening aid and a patient friendly monitoring tool. Device Design The functional requirements are directly derived from stakeholder engagement and are features imposed on the device that must be present from an engineering design boundary condition set. These include: ● Biocompatibility: Materials must be stable upon contact with bodily fluids ● Portability: Size of overall device must be under 250x120x3 mm ● Affordability: Device must be under $25 for the user ● Rapid results: The results must be visible to the user within 30 mins of starting the test ● Low sample volume: Required amount of sample must be under 8 mL ● Shelf stable: Shelf life for at least 6 months ● Precise and accurate: Sensitivity at least 90%, specificity at least 85% ● Accessibility: Must be available over the counter ● Easy to read results: Results are interpreted by the user within 5 mins of trying ● Non-invasive and comfortable: Pain level must be a 1 or below Discussion of Specifications To best suit the needs of the targeted customer, there are functional requirements that the devices should meet. As an improvement to current methods and devices, the devices should remain non-invasive and prioritize patient comfort, be portable for use in outpatient or primary care settings, produce accurate and reliable results within a short timeframe, and have a sufficient shelf life without requiring special storage conditions. Applicable Standards and Codes The proposed designs were evaluated against relevant standards and regulatory considerations. Lateral flow assay components align with International Organization for Standardization (ISO) 13485 for medical device quality management systems [11] and ISO 10993 for biocompatibility of materials contacting biological fluids [12]. Material selection considers Food and Drug Administration (FDA) guidance for over-the-counter diagnostic devices and Clinical Laboratory Improvement Amendments (CLIA) -waived testing requirements for simplicity and safety. Disposal considerations align with standard non-biohazardous household waste classifications for at-home diagnostic tests. Design Progression Figure 1: Design process for the proposed solutions Our design decisioning process is detailed in Figure 1. This begins with identifying a problem and a need. Research is then done to further explore the issue and identify existing solutions and possible shortcomings. Research done during this part supplements the customer and functional requirements that the device must meet in order to meet the needs of the intended customer and be useful. Based on these needs, each member creates a possible solution out of which a final design is chosen to begin prototyping. The solution is tested and ends in a go/no go feasibility decision upon functional testing of the prototype. Design and Analysis of Diagnostic Menstrual Pad The goal of this design is to create a portable, low-cost, and non-invasive biosensing pad capable of detecting biomarkers associated with endometriosis from menstrual blood. The device is designed for at-home testing and provides real-time readouts, serving as an early screening platform to complement clinical diagnostics. The biosensing pad integrates multiple functions: sample collection, filtration, biomarker capture, and signal transduction, into a single layered microfluidic architecture. This reduces the need for additional lab processing steps and external equipment. The proposed system is modeled as a multi-layered pad assembly consisting of a collection layer, filtration/membrane layer, and a detection layer. The collection layer absorbs the bulk of menstrual fluid, filters out larger clots, and guides the sample through a capillary driven flow channel. The microfluidic channels are fabricated using wax screen printing, allowing small analytes such as proteins and inflammatory markers to diffuse through [13]. The detection layer contains functionalized microdomains embedded with antibodies selective for endometriosis-related biomarkers such as CA-125 and miR-28 [14], [15]. Upon analyte binding, these sites generate a colorimetric signal, providing a visible indication of molecular interaction. Figure 2: AutoCAD drawing and dimensions of proposed menstrual pad colorimetric test Figure 2 exemplifies that a key strength of this menstrual pad design lies in its non-invasive and user-centered nature, allowing biomarker detection to occur through a natural and routine process without additional discomfort or complex handling. The integration of microfluidic flow channels improves sample uniformity and reduces user error, while the colorimetric readout enables immediate visual feedback without the need for external readers of power sources. The use of inexpensive and disposable materials makes the design scalable and suitable for over-the-counter production. However, the system faces several limitations. Continuous or variable blood flow during menstruation could alter analyte concentration gradients, affecting test consistency and accuracy. Additionally, cross reactivity between biomarkers or nonspecific adsorption on the pad’s layers could lead to false positives or diminished signal clarity. Finally, ensuring stable antibody functionality and shelf life in a low-cost, disposable format remains a challenge that must be addressed through material optimization and environmental testing. Design and Analysis of Lateral Flow Assay with Antibody Capture Device This design for an endometriosis biomarker collection device is designed with a focus on at-home use and reliable indicators for early stages of endometriosis. Using a similar concept to an at-home pregnancy test, the device uses urine in a lateral flow assay, and strips of color appear as a result indicating if there is a presence of a certain biomarker or not. The novelty in this device is that it has a “capture zone” in which antibody-based capture is used to ensure the specified biomarkers are selected and then amplified in order to be more sensitive for the early detection of endometriosis. Figure 3: AutoCAD and dimensions for lateral flow assay with antibody capture The proposed design in Figure 3 would use VDBP (vitamin D binding protein) and Enolase-1, which is a complementary urinary protein. These analytes were found in studies to be the most consistently elevated and detectable in urinary samples from individuals with endometriosis [16]. The early stage of endometriosis is clinically unspecific and biologically subtle; therefore, the device aims to rectify this problem by focusing on capturing and amplifying biomarkers that are present in low levels in early-stage endometriosis through antibody-based capture. Antibody-based capture was chosen over other binding chemistries (such as aptamers or peptides) because antibodies provide the highest specificity and affinity for these clinically validated targets, allowing reliable enrichment even when analyte levels are extremely low during early disease stages [17]. To support this biology, polyester fiber was selected for the conjugate pad due to its highly consistent release of antibodies, while nitrocellulose was used for the membrane because of its excellent protein-binding capacity and predictable capillary flow, ensuring strong and stable test-line formation. The device housing is made from low-cost polypropylene for durability and manufacturability. Altogether, the choice of biomarkers and antibody-based capture, combined with optimized materials, results in a non-invasive, portable, affordable diagnostic designed to deliver fast, stable, and accurate detection of early-stage endometriosis at home. The strength of this device is that it allows for at-home and accessible collection of biomarkers for the indication of the presence of endometriosis at earlier stages. Although the device is sensitive enough to detect biomarkers related to endometriosis, it is not as accurate as opposed to sending a sample to a lab that can do PCR amplification. Even though the chosen biomarker has promising results to be an indicator of endometriosis, there is yet no definitive biomarker that can singularly detect if a patient has endometriosis at an early stage. This is why this device specifically only aims at being an indicator for a person to follow up more often with a healthcare provider for the risk of endometriosis if the test presents as positive. Design and Analysis of Disposable Lateral Flow Cassette The disposable lateral flow cassette functions similarly to that of a covid test such that it provides a non-invasive tool to potentially detect endometriosis. In addition to being non-invasive, it is low in cost and over the counter. With compact dimensions, the device is portable and enables patients to take the test anywhere rather than waiting for availability at health care facilities to get a diagnostic laparoscopy (the current gold standard for endometriosis detection). The device features a plastic casing as displayed in Figure 4 below with a sample pad, nitrocellulose test strip, and an absorbent pad. The top plastic casing has an inlet that receives the urine sample from the user. This inlet leads to the sample collection pad which is pre-treated to buffer and remove any large particles that may be in the urine. Then, once the sample pad becomes saturated, the filtered urine travels into the test strip through capillary action. The test strip contains antibodies that are potential indicators of endometriosis. Upon contact with the urine, these antibodies rehydrate and bind to the target biomolecules found in the collected sample to then provide a result and control line within thirty minutes. Lastly, to ensure that the results form and show up accurately in the display window, an absorbent pad is placed at the end of the device. This pad absorbs all excess liquid and prevents backflow. Additionally, the device includes instructions on how to ensure proper results along with how to interpret the results. Overall, this lateral flow cassette provides a potential alternative to rapid detection of endometriosis while being low-cost and non-invasive. CA-125, VEGF, and the estradiol/progesterone ratio are the biomarkers that will be on the test strip for endometriosis detection. CA-125 is a glycoprotein that is commonly found in the endometrial tissue, and its over expression in the urine sample is an indicator of inflammation. Endometriosis lesions often get inflamed, and they release CA-125. This is why an elevated level of this biomarker can be used to detect endometriosis in combination with others. VEGF is another protein that is an indicator of new blood vessel growth (angiogenesis). Endometriotic growths require an increased blood supply, so an increase in VEGF can indicate that these lesions are growing and further developing. Lastly, the estradiol/progesterone ratio (E2/P4 ratio) is an indicator of a hormonal imbalance. Women with endometriosis often experience elevated estrogen levels accompanied by insufficient progesterone activity, creating an estrogen-dominant hormonal environment that supports lesion persistence and pain. Estradiol and progesterone metabolites are reliably excreted in urine, enabling non-invasive detection. Together, these three biomarkers cover inflammation, angiogenesis, and hormonal imbalances, which provides a reliable test to detect endometriosis. The different combinations of biomarkers used to provide reliable results, the ability to take the test at any location, and the low-cost design all add to the strengths of the device. However, it is important to stress that this device is a tool to follow up with a medical professional depending on the results— not a formal diagnosis or treatment. Figure 4: AutoCAD and measurements for disposable lateral flow cassette Design and Analysis of Disposable Macro Channel Menstrual Cup The proposed macro channel menstrual cup design integrates a lateral flow assay (LFA), which is what you would find in a pregnancy test, into a menstrual cup collection system. This will enable early, noninvasive detection of endometriosis biomarkers at home. This system has two main components, the reusable menstrual cup and disposable LFA insert component. The Reusable menstrual cup is the collection component. It functions as a biocompatible (made of medical grade silicon), flexible container to collect menstrual effluent. After collection, the user uses the applied disposable insert which is used for endometriosis detection. This is the disposable LFA insert, which is the detection component. It includes a nitrocellulose test strip and sample pad that contains reagents that specifically target certain biomarkers like CA-125. Using a series of filters, the pads filter out particulates which allow fluid to get through conjugate, test, and control zones. In a prompt 10-15 minutes, colorimetric lines indicate the presence or absence of elevated biomarker levels that are associated with endometriosis. Being two parts, the reusable collect part can be sterilized and reused to add sustainability, usability, and affordability. The LFA component remains disposable due to reagent use and to maintain hygiene and prevent cross contamination. The strength of this design is that it is noninvasive and comfortable. Using a familiar menstrual cup design as seen in Figure 5 below, there is less stigma or fear that typically comes with medical sampling. Since this is a familiar menstrual cup integrated with a well-known lateral flow assay (LFA) found in pregnancy tests, it is very accessible for at-home use. Given that it uses LFA, it aids in minimizing costs of this design. The reusability of the menstrual cup allows for reducing costs as well. Given that menstrual cups and LFA are already widely used, it makes the manufacturing process very easy. Figure 5. AutoCAD Drawing of Macro Channel Menstrual Cup Final Design Figure 6. Final product design After careful consideration and review of the functional and customer requirements, Figure 6 presents the final proposed solution that was agreed upon in more detail. The Disposable Lateral Flow Cassette design was chosen, but with modifications made to the biomarkers that the device selects for. This design met all of the criteria compared to the other designs. By using urine as the method of sample collection, the requirements of being non-invasive is met and maximizes comfort for the user. The material choice and straight-forward design keeps the device affordable, compact, accessible, and stable. Choosing to use a multi-panel of biomarkers improves the specificity and sensitivity of the results. Overall, the simplicity of this device is a great asset allowing all of the necessary functional specifications and requirements to be met. Table 1. Bill of materials for prototype Part Description Unit Prototype Cost (est) Cassette top Polypropylene casing with sample inlet and result display window 1 $5.50 Cassette bottom Polypropylene casing with ribs to hold test strip in place 1 $5.50 Test strip Nitrocellulose test strip with control and test lines 1 $0.60 Antibodies Ca-125 VEGF Estradiol/Progesterone ~ 10 µL $10.00 Sample pad Buffered glass fiber pad to filter sample 1 $0.30 Absorbent pad Cellulose pad to absorb excess fluid 1 $1.50 Table 1 presents a bill of materials for the cost of materials during prototyping. The need to create an affordable device plays an important role in what materials are chosen for the design of the device. Risks and Mitigations Biological variability is a significant challenge for non-invasive urine-based screening devices. Biomarker concentrations may vary due to natural factors such as menstrual cycle, dehydration, hormonal contraception, or a concurrent disease. These variations introduce the potential for false negative results during low biomarker expression and false positives when inflammation is unrelated to endometriosis. Additionally, because the proposed device relies on antibody-based detection, there is a tradeoff between sensitivity and specificity, as antibody assays favor sensitivity and may respond to elevated biomarkers that are not exclusive to endometriosis. To mitigate these risks, the final design emphasizes the multi-biomarker detection rather than reliance on a single analyte. Control lines are incorporated to verify the proper assay function. These devices are intentionally positioned as screening tools rather than diagnostic confirmations, which makes increased probability of false positives acceptable. A positive result is intended to encourage timely follow-up with a healthcare provider. Aiming to support earlier clinical intervention rather than serving as the sole diagnosis. Antibody-based detection reagents are projected to remain stable under room temperature (~20℃) storage through lyophilization, a freeze-drying process adhering the antibodies to the nitrocellulose substrate, and the inclusion of stabilizing buffers, consistent with commercially available lateral flow assays. The device is designed to operate within a temperature range consistent with indoor humidity-controlled environments and body-adjacent sampling conditions. Future work will include accelerated aging studies and temperature cycling tests to confirm reagent stability and result consistency. Conclusion As a team, we followed a structured engineering design process to explore multiple non-invasive screening prototypes for endometriosis, guided by functional requirements and literature-supported biomarker selection. This process began with each of the four members designing an individual prototype. A house of quality chart was then used as a metric to determine which of the proposed designs to move forward with in order to finalize the final design. While no experimental validation has yet been conducted, the design analyses demonstrate technical feasibility and clear pathways for future development. The disposable lateral low cassette emerged as the most promising concept based on requirement satisfaction, manufacturability, and user accessibility. Future work focuses on prototyping and benchtop testing to address biological variability and diagnostic reliability. This work aims to build a foundation for future non-invasive at-home screening technologies for endometriosis. By enabling accessible screening earlier in the patient care pathway, these concepts have the potential to reduce the diagnostic delay between symptom onset and surgical diagnosis. Figure 7: House of Quality Summary for an Endometriosis Detection Device Explanation of Scoring: Figure 7 above illustrates the relationship between the customer requirements for a non-invasive diagnostic menstrual pad and the corresponding technical specifications needed to meet those expectations. Each customer requirement, such as biocompatible materials, device cost, fluid volume handling, and sensitivity were evaluated against these needs using a weighted relationship scale (1 = weak, 3 = moderate, 9 = strong). The analysis reveals that material biocompatibility and low device cost are the most influential factors contributing to user safety and affordability, while microfluidic flow control and reagent sensitivity are key to achieving rapid and accurate results. The correlation matrix at the top (roof of house) highlights where design trade-offs occur, for instance, improving sensitivity may increase cost or reduce shelf life.

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