 Good evening, I am Vishaka Mahajan and I am a PhD student at the Liggins Institute. I am interested in studying reproductive biology with a specific focus on the endometrium. Today, I would like to tell you a little bit about tets and their role in the endometrium. Now before I start with tets, I would like to explain what is the endometrium. The endometrium is the innermost lining of the uterus or the womb. It is the only tissue in the human body that has the ability to shed, regenerate and differentiate in a controlled and cyclic manner each month. This unique capacity is a fundamental requirement for a successful pregnancy. However, if there is no pregnancy, this tissue sheds, leaving the body during period and the whole cycle starts all over again. Did you know an average woman spends around 3,500 days of her life menstruating? The menstrual cycle is guided by two main hormones, estrogen and progesterone. These two work in a synchronized manner to degenerate the upper layer, leaving behind a lower layer. This lower layer begins to regenerate in the proliferative phase. This makes up for the first half of the menstrual cycle. The proliferative phase begins upon estrogen influx, which allows the endometrium to grow and prepares it for the second phase, which is the secretory phase. During this phase, progesterone is the dominating hormone and it works with lower levels of estrogen. Progesterone diminishes estrogenic activity and it stops the endometrium from growing, thereby allowing a structural change. This prepares the endometrium to receive an embryo. If there is no embryo or pregnancy, there is a steep decline in both these hormones that triggers the menstrual bleed. Now, there are several mechanisms that are required to maintain these fluctuating hormonal levels and most of these we do not have a very clear understanding of. One such mechanism is epigenetics. Epigenetics literally means above or on top of genetics. This means that these changes do not affect the sequence, but only the confirmation. Let us take a look at this for a minute. This represents DNA that is intricately wrapped around and within parts of this DNA lie your genes. This entire unit is present in every cell of the body. So, then how do your eyes know what they should do or your liver know what it should do? That is because depending on the location, there are certain genes that are active and certain genes that are inactive. Epigenetics work as chemical tags. They attach themselves to certain parts of the sequence, opening the structure, thereby allowing factors to access the gene. These factors tell the gene what they should do, where they should do, why they should do it and how they should do it. If these factors reach the gene, this makes the gene active. In certain cases, these chemical tags tighten the structure, thereby making this gene inaccessible and inactive. Epigenetics is a very dynamic process. This means that these chemical tags can be influenced by environment, lifestyle, diet and hormones. But what makes them interesting is that they are reversible, which means that they can be used as potential targets for drug discovery. Now, there are several ways through which epigenetic marks act and two of which that I am going to be discussing today is DNA methylation and hydroxymethylation. Speaking of DNA methylation, DNA methylation is a process that involves attaching a methyl group to a certain site on the DNA sequence. This tightens the structure making a gene inactive. These are driven by DNA methyltransferases, often known as DNMTs and is often associated with gene silencing. A classic example of DNA methylation is the aguti mice. Just by looking at these two, can you tell that they are identical twins? Aguti is a gene that adds skin color. In one mouse, this gene is active or expressed. This makes this mouse yellowish in color, fatter and more prone to diabetes as well as cancer. Whereas in this mouse, only this gene is inactive. This makes the mouse healthier, browner and leaner. Now, just think about this. If methylation pattern of one gene can do so much to the body, how much does a combined methylation factor of all the genes have on the body? Now, coming back to the endometrium, current literature suggests abnormal methylation patterns are linked with several disease states. DNMTs are known to be overexpressed in the endometrium and in disease states such as endometrial cancer and endometriosis. Since the menstrual cycle is an extremely dynamic process, we wanted to see how DNMTs are expressed throughout it. We found out that DNMTs are upregulated in the proliferative phase or the first half of the cycle. So far, we've understood a little bit about methylation. We know that it is associated with gene silencing. Recently, another epigenetic mark has come to light called hydroxymethylation. This involves attaching a hydroxyl group to the same site on the DNA sequence as methylation targets. Now, structurally, they are very, very similar, but functionally, hydroxymethylation activates gene. So now, there is the DNA sequence and there is one site on the DNA sequence that these two epigenetic tags want to come and target. This makes it really difficult for identification techniques to actually distinguish between these two epigenetic marks, thereby limiting our understanding of this process. Hydroxymethylation is driven by TETs or 10-11 translocation proteins. Currently, there are three known TETs, TET 1, TET 2 and TET 3. These three have been known to play a role in the female reproductive tract, but its exact role in the endometrium remains unexplored. This brings me to the aim of my study, which was to characterize the distribution of TETs throughout the endometrium and to understand the regulation of TETs in endometrial cell types upon exposure to hormones. In order to do that, we divided the study into two parts. For the clinical aspect, we recruited healthy, fertile, reproductive-aged women and extracted endometrial biopsies. These biopsies were then graded depending upon the menstrual cycle phases they were collected in. We then used experimental and statistical tools to determine TET expression throughout the cycle. Since the endometrium consists of two main cell types, epithelial and stromal, we wanted to see how TETs are expressed differentially in these two cell lines. We used healthy endometrial cell lines and treated them individually with estrogen, progesterone and a combined estrogen-progesterone treatment. We then again used experimental and statistical tools to determine TET expression in these two cell lines. The first part of our study revealed that TETs are upregulated or active in the second half of the cycle. This implies that menstrual cycle phases actually influences gene activity and it can also be correlated to the previous data that was DNMTs that drive methylation and are expressed or active in the proliferative phase. Further, we also saw that in cell types, epithelial cells that are predominantly found towards the outer part of the endometrium, TETs were upregulated upon progesterone treatment. Whereas in stromal cells that are found deeper within the tissue, TETs were seen to be upregulated by estrogen treatment. This part of the study highlights the fact that even though the endometrium is one whole tissue, it does contain different cell types that actually behave very differently. Thus concluding, the endometrium is a dynamic tissue that is constantly changing. TETs are not only active or expressed in the endometrium but are also upregulated during the secretory phase. Lastly, TETs are also seen to be expressed differentially in endometrial cell types. So, the take home from this talk is that menstrual cycle phases and cell types are extremely essential covariates that need to be taken into consideration while conducting endometrial studies. In the future, we aim to see how aberrant methylation and hydroxymethylation patterns could contribute to diseases such as endometriosis. I would like to acknowledge my supervisor Dr. Anna and everybody else who was a part of this study and a big thank you to all of you for listening. That would be all.