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Example of gene-variants and whether a patient carries it from 23 and me (Source: dnatestingchoice.com)

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ebb&flo plans to use a similar front end delivery for their patients which will show which gene variants they carry. This can further allow endometriosis detection and susceptibility. Additionally, the gene variants will be useful information for drug-drug and drug-gene interactions

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To increase the database, more research needs to be done. This can include researching more biomarkers that can be used to track endometriosis, more specifically gene indicators. Additionally, for the genes that have been under-researched, they can be researched with a larger cohort study. Lastly, more drug-gene and drug-drug interactions can be researched for both current and future drugs and discovered genes

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As all the research has been collected, the next step would be to create the physical database. For Backend development, Python and SQL would be the best solution as they are the best-known programming languages to organize databases. For frontend development, which includes user interaction and UX/UI development, HTML and Python are most commonly used

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Medications Evaluated in Drug‐Drug Interaction Studies With Elagolix from Polepally et al. (2020)

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Results from drug-drug interaction trial

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Results from drug-drug interaction trial

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Due to the mechanisms of the drug and other drug interactions, the dosage must be monitored.

Elagolix is a substrate for CYP3A; co-administration of elagolix with CYP3A inhibitors, such as ketoconazole, may increase elagolix plasma concentrations.

Solution: Decrease Elagolix dosage

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Orilissa mechanism of competitive antagonism of GnRH receptors at the anterior pituitary gland from Shebley et al. (2020)

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Example Drug-Drug Interaction: Orlissa and Ketoconazole 

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Example Drug-Drug Interaction: Orlissa and Ketoconazole 

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Orilissa (Elagolix)

GnRH antagonist

competes for GnRH receptor occupancy with endogenous GnRH and blocks receptors upon binding

Antagonism

Enters the pituitary and competes with GnRH to sit in the receptor

LH/FSH

Suppresses luteinizing hormone (LH) and follicle-stimulating hormone (FSH)

Estradiol

Subsequent suppression of estradiol levels occurs

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Why are drug-gene interactions important?

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Why are drug-gene interactions important?

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3D model of Fluvoxamine (Source: Wikimedia Commons)

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Alternative solution: Fluvoxamine -> Not metabolized by CYP2C19

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Alternative solution: Fluvoxamine -> Not metabolized by CYP2C19

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Figure 5: CYP2C19 genotype metabolizer speed from Ellis et al. (2009)

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In people who are rapid metabolizers, the CYP2C19 enzyme has high activity. This means that those who are rapid metabolizers break down some medicines quickly and may need different doses or a different medicine. The drug is eliminated too fast and does not achieve a high enough concentration in the blood to be effective.

Citalopram is metabolized by CYP2C19. If someone carried the CYP2C19*17 genotype, Citalopram would not be useful.

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3D model of the Citalopram(Source: Wikimedia Commons)

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Example Drug-Gene Interaction: Citalopram and CYP2C19

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Example Drug-Gene Interaction: Citalopram and CYP2C19

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Citalopram

Brand name: Celexa

Antidepressant of the selective serotonin reuptake inhibitor class

Used to treat major depressive disorder, obsessive-compulsive disorder, panic disorder, and social phobia

CYP2C19 gene

Provides instructions for making an enzyme that is found primarily in liver cells

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The ten genes identified in endometriosis microarray studies. Table from Hansen and Eyster (2010). 

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As ebb&flo's genetic testing kit wants to create a non-invasive solution to endometriosis detection, the database requires genes that should be tested to be able to detect endometriosis. A large reason for endometriosis patients not having genetic testing thus far is due to a lack of research in this field. Not all genes were promising in detecting endometriosis. Many were under-researched or require a large cohort study to be able to confidently be used as a detection gene.

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Explains the relationships studied that are added to the database from the Centers for Control and Disease Prevention

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A pharmacogenomics is the study of how genes affect a person's response to drugs. The database is used for personalising pharmacological therapy by understanding the impact of genetic variability on drug activity. In the case of ebb&flo, the database will be used to determine the best patient care for endometriosis patients and assist with early detecting through genetic testing - a non invasive solution to current methods.

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ebb&flo uses a very similar genetic testing and medicine suggestion platform used by many other companies thus far. Here are some of the companies ebb&flo took inspiration from:

DOTLAB

Adyn

HERA

ZRT Lab

ROSE Endometriosis

23andMe

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Abo C, Biquard L, Girardet L, Chouzenoux S, Just P-A, Chapron C, Vaiman D, Borghese B. 2022. Unbiased in silico analysis of gene expression pinpoints circulating miRNAs targeting KIAA1324, a new gene drastically downregulated in ovarian endometriosis. Biomedicines. 10(9):2065. doi:10.3390/biomedicines10092065. [accessed 2022 Oct 22]. https://www.mdpi.com/2227-9059/10/9/2065/htm.

Bendifallah S, Dabi Y, Suisse S, Jornea L, Bouteiller D, Touboul C, Puchar A, Daraï E. 2022. MicroRNome analysis generates a blood-based signature for endometriosis. Sci Rep. 12(1):4051. doi:10.1038/s41598-022-07771-7. [accessed 2022 Oct 31]. https://www.nature.com/articles/s41598-022-07771-7.

Bendifallah S, Puchar A, Suisse S, Delbos L, Poilblanc M, Descamps P, Golfier F, Touboul C, Dabi Y, Daraï E. 2022. Machine learning algorithms as new screening approach for patients with endometriosis. Sci Rep. 12(1):639. doi:10.1038/s41598-021-04637-2. [accessed 2022 Nov 1]. https://www.nature.com/articles/s41598-021-04637-2.

Chapron C, Lafay-Pillet M-C, Santulli P, Bourdon M, Maignien C, Gaudet-Chardonnet A, Maitrot-Mantelet L, Borghese B, Marcellin L. 2022. A new validated screening method for endometriosis diagnosis based on patient questionnaires. EClinicalMedicine. 44(101263):101263. doi:10.1016/j.eclinm.2021.101263. https://www.sciencedirect.com/science/article/pii/S2589537021005447.

Moustafa S, Burn M, Mamillapalli R, Nematian S, Flores V, Taylor HS. 2020. Accurate diagnosis of endometriosis using serum microRNAs. Am J Obstet Gynecol. 223(4):557.e1-557.e11. doi:10.1016/j.ajog.2020.02.050. https://www.sciencedirect.com/science/article/pii/S0002937820303215.

Senocak GNC, Yapca OE, Yılmaz EPT, Ozturk N, Ozdes S, Kumtepe Y. 2022. May endocan be a new biomarker in the diagnosis of endometriosis? J Gynecol Obstet Hum Reprod. 51(7):102423. doi:10.1016/j.jogoh.2022.102423. https://www.sciencedirect.com/science/article/pii/S2468784722001064.

Sivajohan B, Elgendi M, Menon C, Allaire C, Yong P, Bedaiwy MA. 2022. Clinical use of artificial intelligence in endometriosis: a scoping review. NPJ Digit Med. 5(1):109. doi:10.1038/s41746-022-00638-1. [accessed 2022 Nov 15]. https://www.nature.com/articles/s41746-022-00638-1.

Shebley, M., Polepally, A. R., Nader, A., Ng, J. W., Winzenborg, I., Klein, C. E., Noertersheuser, P., Gibbs, M. A., & Mostafa, N. M. (2020). Clinical pharmacology of elagolix: An oral gonadotropin-releasing hormone receptor antagonist for endometriosis. Clinical Pharmacokinetics, 59(3), 297–309. https://doi.org/10.1007/s40262-019-00840-7

Polepally, A. R., Ng, J. W., Salem, A. H., Dufek, M. B., Parikh, A., Carter, D. C., Kamradt, K., Mostafa, N. M., & Shebley, M. (2020). Assessment of clinical drug‐drug interactions of elagolix, a gonadotropin‐releasing hormone receptor antagonist. Journal of Clinical Pharmacology, 60(12), 1606–1616. https://doi.org/10.1002/jcph.1689

Anastasiu CV, Moga MA, Elena Neculau A, Bălan A, Scârneciu I, Dragomir RM, Dull A-M, Chicea L-M. 2020. Biomarkers for the noninvasive diagnosis of endometriosis: State of the art and future perspectives. Int J Mol Sci. 21(5):1750. doi:10.3390/ijms21051750. [accessed 2022 Nov 29]. http://dx.doi.org/10.3390/ijms21051750.

Breitbach A. Milenia HybriDetect – A universal lateral flow development platform. Milenia Biotec. [accessed 2022 Nov 29]. https://www.milenia-biotec.com/en/universal-lateral-flow-development-tool/.

Pokrovenko DA, Vozniuk V, Medvediev MV. 2021. MicroRNA let-7: A promising non-invasive biomarker for diagnosing and treating external genital endometriosis. J Turk Soc Obstet Gynecol. 18(4):291–297. doi:10.4274/tjod.galenos.2021.07277. [accessed 2022 Nov 30]. http://dx.doi.org/10.4274/tjod.galenos.2021.07277.

Potential RNA markers of abnormal heart rhythms identified in circulating blood. 2018 Mar 19. Medical Xpress. [accessed 2022 Nov 29]. https://medicalxpress.com/news/2018-03-potential-rna-markers-abnormal-heart.html.

Sun D, Wang Y, Wang L, Guo X. 2021. MicroRNA-342 promotes the malignant-like phenotype of endometrial stromal cells via regulation of Annexin A2. Anal Cell Pathol (Amst). 2021:1328682. doi:10.1155/2021/1328682. [accessed 2022 Nov 30]. http://dx.doi.org/10.1155/2021/1328682.

Tucci, F. C., Zhu, Y.-F., Struthers, R. S., Guo, Z., Gross, T. D., Rowbottom, M. W., Acevedo, O., Gao, Y., Saunders, J., Xie, Q., Reinhart, G. J., Liu, X.-J., Ling, N., Bonneville, A. K. L., Chen, T., Bozigian, H., & Chen. (2005). 3-[(2 R )-amino-2-phenylethyl]-1-(2,6-difluorobenzyl)-5-(2-fluoro-3-methoxyphenyl)- 6-methylpyrimidin-2,4-dione (NBI 42902) as a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor. Design, synthesis, and in vitro and in vivo characterization. Journal of Medicinal Chemistry, 48(4), 1169–1178. https://doi.org/10.1021/jm049218c

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Abo C, Biquard L, Girardet L, Chouzenoux S, Just P-A, Chapron C, Vaiman D, Borghese B. 2022. Unbiased in silico analysis of gene expression pinpoints circulating miRNAs targeting KIAA1324, a new gene drastically downregulated in ovarian endometriosis. Biomedicines. 10(9):2065. doi:10.3390/biomedicines10092065. [accessed 2022 Oct 22]. https://www.mdpi.com/2227-9059/10/9/2065/htm.

Bendifallah S, Dabi Y, Suisse S, Jornea L, Bouteiller D, Touboul C, Puchar A, Daraï E. 2022. MicroRNome analysis generates a blood-based signature for endometriosis. Sci Rep. 12(1):4051. doi:10.1038/s41598-022-07771-7. [accessed 2022 Oct 31]. https://www.nature.com/articles/s41598-022-07771-7.

Bendifallah S, Puchar A, Suisse S, Delbos L, Poilblanc M, Descamps P, Golfier F, Touboul C, Dabi Y, Daraï E. 2022. Machine learning algorithms as new screening approach for patients with endometriosis. Sci Rep. 12(1):639. doi:10.1038/s41598-021-04637-2. [accessed 2022 Nov 1]. https://www.nature.com/articles/s41598-021-04637-2.

Chapron C, Lafay-Pillet M-C, Santulli P, Bourdon M, Maignien C, Gaudet-Chardonnet A, Maitrot-Mantelet L, Borghese B, Marcellin L. 2022. A new validated screening method for endometriosis diagnosis based on patient questionnaires. EClinicalMedicine. 44(101263):101263. doi:10.1016/j.eclinm.2021.101263. https://www.sciencedirect.com/science/article/pii/S2589537021005447.

Moustafa S, Burn M, Mamillapalli R, Nematian S, Flores V, Taylor HS. 2020. Accurate diagnosis of endometriosis using serum microRNAs. Am J Obstet Gynecol. 223(4):557.e1-557.e11. doi:10.1016/j.ajog.2020.02.050. https://www.sciencedirect.com/science/article/pii/S0002937820303215.

Senocak GNC, Yapca OE, Yılmaz EPT, Ozturk N, Ozdes S, Kumtepe Y. 2022. May endocan be a new biomarker in the diagnosis of endometriosis? J Gynecol Obstet Hum Reprod. 51(7):102423. doi:10.1016/j.jogoh.2022.102423. https://www.sciencedirect.com/science/article/pii/S2468784722001064.

Sivajohan B, Elgendi M, Menon C, Allaire C, Yong P, Bedaiwy MA. 2022. Clinical use of artificial intelligence in endometriosis: a scoping review. NPJ Digit Med. 5(1):109. doi:10.1038/s41746-022-00638-1. [accessed 2022 Nov 15]. https://www.nature.com/articles/s41746-022-00638-1.

Anastasiu CV, Moga MA, Elena Neculau A, Bălan A, Scârneciu I, Dragomir RM, Dull A-M, Chicea L-M. 2020. Biomarkers for the noninvasive diagnosis of endometriosis: State of the art and future perspectives. Int J Mol Sci. 21(5):1750. doi:10.3390/ijms21051750. [accessed 2022 Nov 29]. http://dx.doi.org/10.3390/ijms21051750.

Breitbach A. Milenia HybriDetect – A universal lateral flow development platform. Milenia Biotec. [accessed 2022 Nov 29]. https://www.milenia-biotec.com/en/universal-lateral-flow-development-tool/.

Pokrovenko DA, Vozniuk V, Medvediev MV. 2021. MicroRNA let-7: A promising non-invasive biomarker for diagnosing and treating external genital endometriosis. J Turk Soc Obstet Gynecol. 18(4):291–297. doi:10.4274/tjod.galenos.2021.07277. [accessed 2022 Nov 30]. http://dx.doi.org/10.4274/tjod.galenos.2021.07277.

Potential RNA markers of abnormal heart rhythms identified in circulating blood. 2018 Mar 19. Medical Xpress. [accessed 2022 Nov 29]. https://medicalxpress.com/news/2018-03-potential-rna-markers-abnormal-heart.html.

Sun D, Wang Y, Wang L, Guo X. 2021. MicroRNA-342 promotes the malignant-like phenotype of endometrial stromal cells via regulation of Annexin A2. Anal Cell Pathol (Amst). 2021:1328682. doi:10.1155/2021/1328682. [accessed 2022 Nov 30]. http://dx.doi.org/10.1155/2021/1328682.

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1) Introduction, Kit Development, Drug-Drug Interactions, Future Outlook - Abidur
2) Introduction, Future Outlook, Kit Development (biomarker selection & figures) - Shuha
3) Biomarker selection, Researching Drugs, Gene-Drug Interaction, Future Outlook - Rubaina

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1) Producing antibodies for at least some of our biomarkers
2) Comparing different dyes with gold nanoparticles, so the best option is used to label our biomarkers.
3) Trying out different flow rates of serum and pad sizes
4) Comparing a prototype of our kit's accuracy with clinical standards (other flow assays, machine-learning based algorithms, laparoscopy)

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We recently reached out to Alana Ogata, a professor in chemistry at the University of Toronto (Mississauga) who works in diagnostic pipelines for gynaecological diseases.
She has expressed interest in further discussions and potential collaboration!

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Source: Pokrovenko, D. A. et al. MicroRNA let-7: A promising non-invasive biomarker for diagnosing and treating external genital endometriosis.

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MicroRNA let-7b as a diagnostic biomarker

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MicroRNA let-7b as a diagnostic biomarker

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Two studies demonstrated let-7 family is dysregulated in the serum f endometriosis patients.
Let-7 promotes cell differentiation and may act as a tumor suppressor. Meanwhile, let-7b is associated with cyclin D1, an estrogen-regulated gene involved in cell proliferation.
Another study showed the downregulation of let-7, which targets the KRAS gene.
The loss of this miRNA may initiate the progression of ectopic lesions. It may represent a link between inflammation and tumor-like growth in endometriosis.

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Source: Sun, D. et al. MicroRNA-342 Promotes the Malignant-Like Phenotype of Endometrial Stromal Cells via Regulation of Annexin A2

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Selecting miRNA biomarkers for diagnostic panel

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Selecting miRNA biomarkers for diagnostic panel

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Let-7b: expression levels found to be significantly lower in endometriosis patients than in controls.
miR-451a: expression shown to be decreased in eutopic endometrial tissues taken from endometriosis patient.
miR-342-3p: higher levels shown to reduce the metabolic gene expression in adipocytes of endometriosis patients

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The Future of Endometriosis : miRNA detection?

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The Future of Endometriosis : miRNA detection?

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The study by Bendifallah et al found 86 miRNAs

10 of them have the greatest diagnostic potential, with miRNA 124-3p having been previously associated with endometriosis

most accurate signature provides a sensitivity of 96.8% and specificity of 100%

robust enough to replace the gold standard - diagnostic surgery

Recent studies, including one by Qui et al. used CRISPR-Cas9 combined with horseradish peroxidase to detect miRNAs in a very low-cost manner. This could be the future of endometriosis detection.

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While patients are prescribed many medications, they are not fully aware of side-effects with co-administered drugs e.g. Advil for pain.
Ebb&flo believes in full-transparency and patient empowerment and one of our sub-projects was to create a drug-drug interactions database.

We highlight the clinical trial of one such drug Elagolix(Orlissa) and its drug-drug interactions.

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Drug-Drug Interactions with Common Endometriosis Medications

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Empowering patients with our pharmacogenomic databases 

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Empowering patients with our pharmacogenomic databases 

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Generating antibodies in animals or cell lines is a very expensive and laborious process, with a variety of ethical concerns.

Recent research has shown how we can produce over 70 different types of full-length antibodies in a specific E.coli strain called SHuffle (Robinson et al., 2015). This strain is equipped with a modified oxidative cytoplasm, so we can have disulfide bond formation and thus proper folding and conformation of the antibodies. The strain also has extra chaperones (e.g.  GroEL/S systems) to guide this folding process (Lobstein 2012 et al., 2012). (Robinson et al., 2015)

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Th iGEM kit involves 2 components :
1) A lateral flow assay (LFA)
2) Production of antibodies for the lateral flow assay

Design Principle :
Initially described in 1960 and now widely used, a lateral flow assay detects the presence of target proteins (in our case, endometriosis-related biomarkers) in a liquid sample (in our case, serum from patients) . At the sample pad, where the separated serum is applied, we have a fixed concentration of labelled primary antibodies. These primary antibodies are either labelled with dyes or gold nanoparticles. As the sample flows to the end (where the test strip is), the labeled primary antibodies bind to the biomarkers. Once our samples reach the test line, the target molecules bound to another secondary, immobilized antibodies. Free labeled primary antibodies can continue to travel down the strip to a control line (Sajid et al., 2015).(Teerinen et al., 2014).

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SerumDPT provides real-time separation of serum from plasma in a low-cost manner without using centrifuges.

The SerumDPT tube has two evacuated chambers and the blood enters the collection chamber through a flow regulator. This chamber also houses a micro-fibres-based filtration system that separates cells and clotting factors from the blood.

SerumSTAT delivers a minimum of 250 microlitres of serum from 2mL of drawn blood in a minute.

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Figure 2: A list of drug-gene interactions present within endometriosis treatments

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Step 3: Researching Gene-Drug Interactions

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Step 3: Researching Gene-Drug Interactions

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One of the goals of ebb&flo is to be able to give patients a list of suggested treatments that is tailored to their own biology. When ebb&flow sends out a genetic testing kit, they are not only searching for Endometriosis susceptibility through biomarkers, they are also looking at other genes that might affect the way one's biology reacts to treatment.

Through advance literature research, our group went through every medication we had sourced from patient forums and literature reviews in Step 2 and researched their chemical compositions. From there, the drugs were researched to see whether any particular genes caused additional side effects or simply did not work for a person. This allows us to have a comprehensive list of genes and gene variants ebb&flo would need to test in order to suggest the best possible solution for a patient.

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We went through research papers and patient forums to learn more about the different types of medications endometriosis patients are prescribed or take. Medication for Endometriosis patient include contraceptives, Hormones and Antineoplastics, Progestins, Sex Hormones, Gonadotropin Releasing Hormones, Antigonadotropic agents, and Gonadotropin-releasing hormone antagonists.

Here, we present a few drugs endometriosis patients might take. The ones stated are a current list of the most common Hormones and Antineoplastics taken

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We decided to select serum separated from peripheral blood as our ideal sample for 4 reasons:
1) Almost all of our biomarkers are studied in serum samples.
2) Past studies show that pro-inflammatory cytokines e.g. IL‐6, IL‐1β are significantly different and higher in menstrual blood versus peripheral blood.
3) It is more invasive & expensive (e.g. menstrual cups, tampons) to extract menstrual blood.
4) Hormones and biomarkers need to pass
the salivary gland membranes to be detected.

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Our Kit Development Process : Nifty bacteria, old antibodies and a tale of microRNA power

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Our Kit Development Process : Nifty bacteria, old antibodies and a tale of microRNA power

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Introduction : We are failing patients with endometriosis

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Introduction : We are failing patients with endometriosis

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As we went through our literature review, we came across 2 lines of research : 1) classical biomarkers like the IL-6 cytokine 2) recent bank of research focusing on microRNAs in reliably detecting different forms of endometriosis.

Here, we present a list of the biomarkers, common sources to obtain them from the human body, and the sensitivity* and specificity** of the biomaker.

*Sensitivity percentages reflect the ability of a test to correctly identify patients with a disease

**Specificity percentages reflect the ability of a test to correctly identify people without the disease

More info:

IL6: inflammatory marker successful in detecting endometriosis

IGFBP-1: altered levels successful in detecting ovarian endometriosis

CA125: significantly higher levels due to endometrial tissue growth outside the uterus in endometriosis patients

For their potential in the identification of endometriosis in participants with inflammatory symptoms, 3 biomarkers with elevated serum levels chosen -

IL-8

IL-1B

TNF-a

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The literature review for endometriosis-related biomarker selection had 3 selection criteria:
1) The study needs to report the specificity & sensitivity of the biomarker clearly.
2) The study cannot focus on only 1 type of endometriosis.
3) The study should use either serum or menstrual blood as its initial samples, as biopsy or tissue-based samples would be similar to laparoscopy.

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1) Find appropriate biomarkers for endometriosis
2) Find an appropriate biological sample that covers these biomarkers and a standard way to collect it
3) Incorporate a standard assay to detect these biomarkers

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ebb&flo : A revolutionary end-to-end approach to endometriosis care 

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ebb&flo : A revolutionary end-to-end approach to endometriosis care 

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Seeing how millions of women are being failed by current care approaches, Kelly Goldfeder founded ebb&flow -
a health & biotechnology company that provides marginalized groups with personalized care from chronic diseases, including endometriosis.

Our first project looked at creating a low-cost, accessible & accurate diagnostic kit for endometriosis.

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The Ongoing Challenges with Endometriosis

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The Ongoing Challenges with Endometriosis

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This delay is due to 3 key challenges :
1. Very heterogeneous symptom presentation
2. Great overlap with other diseases (e.g. pelvic pain with ovarian cysts)
3. The lack of accessible diagnostics as laparoscopy can cost about $5000 and is highly reliant on surgical skills & equipment access.

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190 million of women and girls suffer from endometriosis globally and there is an average delay of 6-12 years from having symptoms to getting diagnosed.

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Figure 1 : The endometrium & aberrant endometrial cells on the ovaries and fallopian tubes

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Endometriosis is a systemic & chronic inflammatory disease where endometrial cells grow outside the uterus.

Classical symptoms include pain during periods & sexual intercourse, chronic pelvic pain, nausea, fatigue, and sometimes depression, anxiety, and infertility.

The golden standard of diagnosis and treatment is Laparoscopy - a surgical procedure to remove the aberrant lesions.