Metabolic Regulation Differences Between a Thyroidectomized Mouse and in a Hypophysectomized Mouse

Taking a look at the thyroid gland, as part of the hypothalamic-pituitary-thyroid axis, the two thyroid hormones it releases, triiodothyronine (T₃) and thyroxine (T₄), play an important role in regulating the body’s metabolism. Metabolism is the summation of both anabolic and catabolic chemical reactions that occur in the cells of the body. The more thyroid hormones that have the potential to bind to specific cells, the higher the rate of metabolism that those cells will undergo. Of course, the amount of these hormones needs to be homeostatically regulated under a negative feedback system by the hypothalamus.

Through a humoral control system, the hypothalamus regulates the thyroid hormone concentration in the blood through a specified set point. If either the concentration of the thyroid hormones is too low or too high in the blood, the hypothalamus will increase or decrease, respectively, its release of thyrotropin-releasing hormone (TRH) to the adenohypophysis. Subsequently, the adenohypophysis has cells that respond to specific hormones that the hypothalamus releases, in this case it is TRH. When TRH is present, the anterior pituitary will release more thyroid-stimulating hormone (TSH) which will arouse the thyroid gland to release thyroid hormones. The aim of this study is to observe and interpret the effects on the metabolic activities of mice that either have the absence of the thyroid gland or the absence of the pituitary gland when exposed to three injections that contain T₄, TSH, and propylthiouracil (PTU). Each of those substances possesses an effect on either the hypothalamus/pituitary or the thyroid gland.

Methods

Measuring the Baseline Metabolic Rate (BMR) Through Oxygen Consumption

 This experiment utilized three mice with identical age and sex and of similar weight. One of the mice underwent thyroidectomy, the other underwent hypophysectomy, and the third untouched to become the control subject. They were kept is three separate cages, consuming the same amounts of food and water at the same time each day. A week after baseline measurements, each mouse was individually placed in a sealed chamber that contained a scale for one minute.

This chamber contained two tubes connected. One tube was accessible to the outside air, which will be open when the subject is not being measured, and closed when the subject is being measured, while the other tube ran through a T-shaped pipe, which directed air flow into one of the two passageways. One of the dividing tubes led to a U-shaped manometer filled with water. The manometer measures the oxygen consumption by offsetting the water level of one side of the U to the other side. The other dividing tubes led to a syringe filled with air. This syringe was used to pump air into the tubing system towards the manometer in order to level the water. The amount that the syringe measures when the manometer reaches the original leveled state is the amount of oxygen that the test subject consumed in that one minute.

Each mouse was untreated and then measured for their weight (in kilograms) and oxygen consumption (in milliliters) to establish baseline values. Afterward, each mice was injected with T₄ then remeasured for their weight and oxygen consumption. This was repeated for the administration of TSH and again for the administration of PTU after all three of the mouse had returned to near their standard BMR. Calculations must be made using the oxygen consumption and weight recorded for each mouse after each of the trials in order to calculate the BMR of each mouse in the experiment. The equation is as follows: milliliters of oxygen consumed per hour divided by the weight of the mouse in kilograms. Consequently, the BMR values will be expressed in ml O₂/hr/kg.

Results

Discussion

The Hypothalamic-Pituitary-Thyroid Axis

As mentioned, through a homeostatically regulated negative feedback system, the hypothalamus is the main control center in regulating thyroid hormone release. Nillni (2010) states that the afferent neurons that play an important role in informing TRH neurons in the hypothalamus are the catecholamine neurons, which originates in the brain stem, and the arcuate nucleus of the hypothalamus (ARC). Peripheral neuronal signals that are stimulated for signs of starvation or illness cause a response from the ARC while temperature decrease sensed by peripheral afferent temperature receptors cause a response from the catecholamine neurons. When stimulated, these afferent connections signal the hypothalamus TRH neurons to release more TRH.

Going into context with the negative effects of thyroid hormones on the TRH neurons, in another study by Mariotti and Beck-Peccoz (2016) stated that specialized ependymal cells identified as tanycytes, located along the blood-brain barrier of the choroid plexus as well as the third ventricle, withdraw T₃ and T₄ from the bloodstream across the blood-brain barrier and from the cerebrospinal fluid (CSF). The T₄ will then be converted into the more bioactive hormone T₃, using the type 2 iodothyronine deiodinase (D2) enzyme – which the differences between the two thyroid hormones will be discussed later. As a steroid hormone, T₃ is a hydrophobic hormone that binds mainly on intracellular receptors meaning that it can usually pass through the target cell’s phospholipid bilayer, but in order to enter the TRH neurons it requires a series of active transporters, mainly two: organic anion transporting polypeptide (OATP) and the monocarboxylate transporter type 8 (MCT8). How TRH neurons work as their own differentiated neuron is in the concept of its TRH production. Along the cell’s genome lies the promoter region for the creation of mRNA strands to synthesize TRH to release to the adenohypophysis. Normally, there is an abundance of CREB (cAMP response element binding protein), a transcription factor that, in this case, increases the transcription of the TRH gene into mRNA for translation. T₃ negatively affects this process by binding into thyroid hormone receptor/T₃ retinoid X receptor complex which ultimately lowers the CREB concentration, lowering the transcription of the TRH gene.

As TRH is synthesized by the TRH neurons, the TRH neurons release the hormone through their axon terminals in the median eminence, which is located on the superior side of the adenohypophysis, and into the blood circulation of the adenohypophysis. Specialized cells named thyrotrophs own TRH receptors for the TRH neuropeptide hormone. These TRH receptors are G-protein-coupled receptors. As a result of the G-protein activation, the alpha subunit from the G-protein then activates the protein lipase C enzyme, which, in effect, breaks down phosphatidylinositol 4,5-bisphosphate (PIP₂), which is rich in the cell cytosol as an important substrate to many signaling proteins, to inositol trisphosphate and diglyceride. Inositol triphosphate then causes a release of calcium ions (Ca²⁺) from intracellular stores while diglyceride activates yet another enzyme, this time called protein kinase C. Through these cascade of events that have been amplified by the TRH receptors, the thyrotrophs are induced to activate TSH gene transcription, synthesizing more TSH, and then releasing them into the  hypophyseal portal system in order to stimulate the thyroid gland. Other notable factors that regulate TSH synthesis and secretion are estrogen and testosterone serum concentration, other transcription factors such as Pit-1, and cAMP concentration inside the cell (Mariotti & Beck-Peccoz, 2016).

Not only do thyroid hormones play a role in inhibiting TRH synthesis in TRH neurons of the hypothalamus, but they also play that same specific role in inhibiting TSH creation among the thyrotrophs of the adenohypophysis. In the presence of thyroid hormones T₃ and T₄, the amplification system activated by protein lipase C become blocked, conclusively preventing the synthesis of TSH. 

As TSH moves along the body’s cardiovascular system, the TSH peptide hormone will eventually bind to TSH G-protein coupled receptors on the basolateral membrane of thyroid follicular cells and release thyroid hormones T₃ and T₄. The process of how the thyroid gland synthesizes those hormones will be discussed in this paragraph. On average, adult humans intake of iodine about 150 µg/day. Based on a study by Rousset, Dupuy, Miot, and Dumont in 2015, the iodine that is consumed then become iodide (I^–) before entering the mesenteric bloodstream, becoming absorbed from serum by the thyroid gland. How the thyroid cells (thyrocytes) absorb iodide is attributed to sodium/iodide symporters (NIS) found along the basolateral cell membrane. This active transport pump pumps two sodium ions (Na⁺) down its concentration gradient and into the cell, transporting one I^– against its concentration gradient. The constant concentration gradient of Na⁺, which drives the uptake of I^–, is due to the continuous activity of active sodium/potassium pumps, pumping three sodium ions out of the cell and two potassium ions back into the cell. Subsequent to I^– intake, the I^– ions are then transported across the cytosol and passively transported across the apical side of the cell into the minute cavities of the gland itself filled with a fluid called colloid. This transportation of iodide into the colloid is presumably connected to a transporter protein named pendrin, which transports an iodide ion out of the apical side of the cell, then transports a chloride ion from the colloid into the cell.

In order to begin the synthesis of these hormones, thyroglobulin (Tg) is needed as an important protein that becomes the backbone of thyroid hormones. After mRNA translation of the TSH gene from the nucleus, the protein then undergoes glycosylation – the attachment of glycans to proteins – and dimerization in the rough endoplasmic reticulum, and then final modification by terminal glycosylation at the Golgi apparatus. After final processing, the Tg proteins are packed inside of vesicles, which eventually endocytose Tg proteins into the colloid-filled cavities of the thyroid gland (Rousset et al., 2015)

In the colloid, hydrogen peroxide and the enzyme thyroperoxidase (TPO) readily oxidizes two iodide molecules to form a single iodine molecule, preparing it to bind to tyrosine residues called tyrosyl in Tg proteins in a process called iodination. When an iodine molecule is covalently bound to a tyrosyl of the Tg protein, the resulting complex is called monoiodotyrosine (MIT). Subsequent reactions that iodinate two iodine molecules to a tyrosyl, or adding an additional iodine molecule to MIT, result in diiodotyrosine (DIT). In order to form the two known thyroid hormones, T₃ and T₄, TPO then iodinates neighboring MIT and DIT complexes, through the process of conjugation, together which creates T₃, T₄, or rT₃ (inactive, reversed structural form of T₃), all of which are still bound to the Tg protein peptide backbone. The orientation of the tyrosyl rings is important in determining whether the tyrosyl complex is classified as a T₃ hormone or a rT₃ hormone. If the resulting complex created includes an MIT complex connected to the Tg backbone as the inner ring and a DIT complex connected to the MIT complex as the outer ring, the complex is classified as rT₃ after the process of deiodination. Consequently, if the resulting complex created includes a DIT complex as the inner ring and an MIT complex as the outer ring, the complex is classified as the active T₃ after the process of domination. The large protein complex will then be stored inside the colloid until the needed release of thyroid hormones activated by TSH (Rousset et al., 2015).

When TSH eventually binds to thyrocytes through a G-protein coupled receptor, the resulting cascade of events activates the endocytic receptor on the apical membrane of the cell called megalin. Activation of the megalin protein allows the iodinated Tg to bind to the receptor in order to become endocytosed in the thyrocyte. After internalization, lysosomes containing proteolytic enzymes fuse to the vesicle in order to free T₃, T₄, and rT₃ from their Tg backbone. Following this cleavage, the vesicle is then transported to the basolateral cell membrane of the thyrocyte, releasing the thyroid hormones into the cytosol where they will be transported into the blood circulation through the monocarboxylate transporter protein (Kleinau, et al., 2017; Refetoff, 2015).

Due to their lipophilic structure, thyroid hormones do not travel well in blood by itself, due to water being 92% of blood composition. For this reason, these hormones need to be bound to transport proteins. Only about 0.04% of T₄ and 0.3% of T₃ present in the serum are unbound and free. 70% of both hormones are bound to thyroxine-binding globulin (TBG), 20% are bound to thyroxine-binding prealbumin (TBPA), and 5% are bound to human serum albumin (HSA). Although HSA is much more highly concentrated than the other two transport proteins, TBG’s affinity to the iodothyronines is more than 7000 times stronger than HSA and 50 times stronger than TBPA. The location of the production of these transport proteins varies. TBG is mainly produced in the liver. TBPA can be produced by the liver, kidney, and even parts of the brain’s meninges and ventricles. Lastly, HSA, which occupies more than 50% of the total serum proteins, is also produced in the liver (Refetoff, 2015). 

The general metabolism of iodothyronines takes place in different areas of the body. Three different types of deiodinases are known to have different functions. Type 1 deiodinase (D1) is mainly found in the liver, kidneys, and thyroid. This enzyme’s main function is to increase T₃ levels in the blood by deiodination T₄ hormones as well as clearing any inactive rT₃. Type 2 deiodinase (D2) can be found in the central nervous system, pituitary, skeletal muscles, and brown adipose tissue, maintaining the levels of T₃ in the serum. D2 is also present and active within cells to convert T₄ into T₃ in order to activate any cell-specific physiological changes. Finally, type 3 deiodinase (D3) is only found in the brain. The purpose of D3 solely on regulating the active T₃ concentration by deactivating T₃ into rT₃ (Peeters & Visser, 2017).

In a study performed in 2014 by Mullur, Liu, and Brent, the mechanism of action that these iodothyronines play on targeted cells is very similar to their effects on the TRH neurons discussed in the earlier paragraphs. Once the hormones reach their target cell, they will enter the cell through the same membrane protein as they used to exit the thyroid follicular cells, the MCT8. The other MCT protein is the type 10 variant which is to yet to be examined on its true functionality as MCT8 proteins deal with iodothyronine transportation across cell membranes. It should also be noted that MCT8 proteins have a stronger affinity to T₃ than to T₄. Studies have shown that MCT8 protein deficiencies had led to abnormally high serum T₃ concentrations, while MCT10 deficiencies do not have an impact on T₃ serum levels. Once inside the cell membrane, T₃  hormones will bind to the thyroid hormone receptor/T3 retinoid X receptor complex at a specific promoter region of the specific cell, inhibiting or stimulating the transcription of certain genes within the chromosomes. As for T₄, any T₄ hormones that are transported into a cell will then be deiodinated by type 2 deiodinase, splicing of one of its iodine molecules from the tyrosyl base, ultimately becoming the more bioactive T₃. In particular, genes that regulate basal metabolic rate and development will usually become activated more than repressed. These genes allow for the creation of specific enzymes for fat, carbohydrate, and protein metabolism for the process of gluconeogenesis. Speaking about more specific influences of thyroid hormones, during neurological development, thyroid hormones stimulate the genes that are associated with myelination, neuron differentiation, and neuronal signaling. For skeletal development, the genes activated are associated with bone development and growth (Bassett & Williams, 2016). Lastly, muscle development is especially enhanced during embryonic development and postnatal development all through the influences of thyroid hormones.

Situational Analysis of the HPT Axis among Mice

The dependent variable that was recorded in the experiment was BMR, specifically ml O₂/hr/kg. The reason for using oxygen consumption to record BMR lies in the production of ATP within the body’s cells. Generally, most metabolic reactions require the hydrolysis of ATP in order to acquire energy to drive such reactions. The process of cellular respiration requires oxygen in order to accept terminal electrons from the electron transport chain. If in a situation where BMR is increased due to chemical cellular messengers such as the iodothyronines, more ATP will be generated to supply the cells with the pool of energy. Therefore, oxygen consumption is positively correlated with BMR, hence, its usage in the experiment.

Taking a look at the results of the experiment, the normal mouse reacted normally to all of the injections as expected with an intact HPT axis. Our baseline, standard metabolic consumption recorded from the normal mouse with a value of 1710 ml O₂/hr/kg. For the thyroidectomized (Tx) mouse, under normal conditions, it had metabolic consumption of 1471 ml O₂/hr/kg, which is about 86% of the metabolic consumption of the normal mouse. This can be justified by acknowledging that the subject’s thyroid gland is missing. There will be no iodothyronines in the serum to regulate metabolism. It can also be assumed that there will be a high concentration of TSH within the blood as the TRH neurons will not become inhibited by the presence of T₃, inducing them to release more TRH, which will then stimulate the thyrotrophs to release more TSH. This will be a waste of cellular resources and energy in making TRH and TSH in order to stimulate a nonexistent thyroid gland. Subsequently, the hypophysectomized (Hypox) mouse also showed a metabolic rate that was 87% of the normal mouse. This to be expected in subject three as well. Without a pituitary gland, the subject cannot regulate the production of iodothyronines. In this situation, little to no thyroid-relevant activity should be analyzed as there will be very little T₃ to stimulate the TRH neurons to release TRH. With the absence of the adenohypophysis, no TSH will be produced to induce the thyroid to release any thyroid hormones. Because of this, we can see an abnormally low BMR from subject three.

Moving on to the first trial; the administration of thyroxine in the subjects’ bloodstream. It is noticeable that all of the subjects’ BMR increased. The control subject’s BMR increased by about 10.8% while the Tx and Hypox mice increased by about 25.2% and 23.3%, respectively, from their original recorded BMR. It was expected to see all three of the subjects’ BMR to increase as T₄ can act in increasing metabolic reactions within cells through the already present deiodinases located all around the body. Furthermore, subject one’s BMR did not increase as much as the other subjects. The reasoning behind this could be from the fact that the introduction of T₄ into the subject activated the negative feedback system of the HPT axis, not allowing the influx of T₄ create as big of an effect to BMR as the other subjects. On the other hand, subjects two (Tx mouse) and three (Hypox mouse) experienced larger increases to their BMR than the control mouse most likely due to the current state of each subject. Both subjects were already below the set point of iodothyronine concentration in the serum. For this reason, the injected T₄ had a larger effect on these subjects as their bodies were most likely attempting to compensate for the period of low BMR and iodothyronine concentration.

In trial two, however, only the control mouse and the Hypox mouse experienced a significant increase in BMR when injected with supplementary TSH. Subject one’s BMR increased by 9.8% while subject three’s BMR increased by 24%. The TSH administration was able to act on the thyroid glands of the control mouse as well as the Hypox mouse, stimulating the release of T₃ and T₄, ultimately increasing the metabolism in various parts of their bodies. Again, the larger increase of metabolism that the Hypox mouse experienced can be attributed to the current deficit in metabolic activities within the body, allowing for the larger effect on BMR. On the contrary, subject two’s BMR increased only by 1.4% which is not significant enough to consider the TSH injection effective. Due to the absence of a thyroid gland, subject two’s body was not able to produce and release any iodothyronines to increase metabolic functions. The sole effect of the administration of TSH on the Tx mouse was merely increasing the concentration of TSH in the serum but with no active interaction of the peptide hormone with any gland or cell. 

For the final trial, each mouse was injected with propylthiouracil (PTU). It is known that PTU owns two methods of inhibiting iodothyronine synthesis within the thyroid. ONe mechanism of inhibition PTU exhibits is the interference of the thyroperoxidase (TPO) enzyme located in the colloids of the thyroid gland. Inhibiting TPO results in the termination of the conversion of iodide to iodine. As stated above, the formation of iodine facilitates its binding onto the tyrosyl branches of thyroglobulin. Reducing iodine concentration within the colloid will decrease the creation of iodothyronines within the thyroid gland, ultimately decreasing iodothyronines within the serum and decreasing BMR. The other mechanism of inhibition PTU displays is the inhibition of the D2 enzyme; the enzyme that splices of an outer ring or inner ring iodine in order to transform T₄ into T₃ or rT₃. This will lead to a decrease in the secretion of active T₃ in the bloodstream, influencing overall regulated T₃ serum concentration, leading to a decrease in BMR. It was observed that only the control mouse experienced a decrease in BMR from its standard rate, while the Tx and Hypox mice did not experience a decrease in their BMR. The BMR of the control mouse decreased by 15.9%. Subject two, on the other hand, does not have a thyroid gland for PTU to inhibit, so, it was expected that the subject’s BMR did not decrease. As for the Hypox mouse, although the subject still possesses a thyroid gland, the missing adenohypophysis implies that the subject’s thyroid gland was not being previously stimulated to release iodothyronines to regulate metabolism. So, the PTU administration had no effect on BMR as the thyroid gland is not secreting any hormones in the first place.

Medical Conditions Caused by a Malfunctioning HPT Axis

 Hyperthyroidism is a condition in which the thyroid gland secretes an excessive amount of thyroid hormones in the body. A notable disease which involves hyperthyroidism is Graves’ disease – an autoimmune disorder in which the binding immunoglobulin antibodies on the thyroid actually stimulates the gland’s G-protein coupled receptors, signaling the gland to release more iodothyronines. Due to the heightened metabolic activity, patients with this disease may experience anxiety, hand, tremors, heat sensitivity, weight loss, and enlarged thyroid. Serious problems may also  Currently, doctors and scientists do not know what specifically causes the immune system to suddenly creates antibodies that attack the body’s own tissues, but some are leading towards to the idea of the influence of viral virions on the genetic material of cells. Since the thyroid gland is stimulated to release more hormones than normal, it is anticipated to see an increased uptake of iodine by the thyroid gland. For this reason, one of the diagnostic tests used to diagnose hyperthyroidism is the radioactive iodine uptake test. One treatment option is radioiodine therapy. Patients that undergo this treatment consumes radioactive iodine-131 by pill or liquid. The absorbed radioactive iodine-131 could only be absorbed by the thyroid gland, allowing the substance to destroy a specific amount of follicular cells of the thyroid gland until the patient possesses normal levels of thyroid hormone concentration. Another effective treatment for hyperthyroidism is the use of antithyroid medications such as PTU to inhibit the production of iodothyronines.

 Another disease involving flaws within the HPT axis is hypothyroidism, which consists of a reduction in the activity of the thyroid gland. Hypothyroidism can be caused by a number of situations. For example, having an autoimmune disorder called Hashimoto’s thyroiditis, taking too much hyperthyroidism treatment such as radioactive iodine or anti-thyroid medications, or undergoing the surgical procedure of removing the thyroid gland. Health problems that may be associated due to the lowered regulation of thyroid hormones include damage to peripheral nerves, infertility, and myxedema, which can be explained due to the lack of thyroid hormones to regulate the required metabolic activities the body regularly performs. In order to properly diagnose hypothyroidism, a physician may need to perform blood tests, examining the levels of TSH as well as T₄ in the blood. High levels of TSH with low levels of thyroid hormones may indicate that the thyroid gland is not as active as it should be. As for treatment options, the standard treatment is through the use of synthetic thyroid hormones, such as levothyroxine, which are taken daily in order to return the body to normal euthyroid activity. 

References

• Nillni, Eduardo A. “Regulation of the hypothalamic thyrotropin releasing hormone (TRH) neuron by neuronal and peripheral inputs.” Frontiers in neuroendocrinology vol. 31,2 (2010): 134-56. doi:10.1016/j.yfrne.2010.01.001
• Maria Izabel Chiamolera, Fredric E. Wondisford, “Thyrotropin-Releasing Hormone and the Thyroid Hormone Feedback Mechanism.” Endocrinology, Volume 150, Issue 3, 1 March 2009, Pages 1091–1096,
• Mullur, Rashmi et al. “Thyroid hormone regulation of metabolism.” Physiological reviews vol. 94,2 (2014): 355-82. doi:10.1152/physrev.00030.2013
• Refetoff S. “Thyroid Hormone Serum Transport Proteins.” [Updated 2015 Jun 7]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK285566/
• Pirahanchi Y, Jialal I. Physiology, Thyroid Stimulating Hormone (TSH) [Updated 2019 Apr 25]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK499850/
• Kleinau, Gunnar et al. “Structural-Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work.” Frontiers in endocrinology vol. 8 86. 24 Apr. 2017, doi:10.3389/fendo.2017.00086
• Brent, Gregory A. “Mechanisms of thyroid hormone action.” The Journal of clinical investigation vol. 122,9 (2012): 3035-43. doi:10.1172/JCI60047
• Cheng, Sheue-Yann et al. “Molecular aspects of thyroid hormone actions.” Endocrine reviews vol. 31,2 (2010): 139-70. doi:10.1210/er.2009-0007
• Peeters RP, Visser TJ. “Metabolism of Thyroid Hormone.” [Updated 2017 Jan 1]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK285545/
• Mariotti S, Beck-Peccoz P. “Physiology of the Hypothalamic-Pituitary-Thyroid Axis.” [Updated 2016 Aug 14]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK278958/
• Rousset B, Dupuy C, Miot F, et al. “Chapter 2 Thyroid Hormone Synthesis And Secretion.” [Updated 2015 Sep 2]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK285550/
• Mumtaz, Malik et al. “Radioiodine I-131 for the therapy of graves’ disease.” The Malaysian journal of medical sciences : MJMS vol. 16,1 (2009): 25-33.
• Mayo Clinic. Mayo Foundation for Medical Education and Research (MFMER), https://www.mayoclinic.org/. Accessed 12 May, 2019