Identification of key transcriptional and functional differences in adenomyotic lesions to determine novel therapeutic targets

In the UK, 1 in every 5 women suffer with heavy and painful periods. This has a huge effect on their lives, with many women having to miss work, social and sporting activities. This has a major impact on the economy and the health and wellbeing of women in our country.
A common cause of heavy painful periods is adenomyosis, a condition in which the cells normally found in the inside lining of the womb (called the endometrium), grow abnormally deep in the muscle layer of the womb (called the myometrium). It is estimated that 1 in 10 women suffer from this condition with numbers steadily increasing. In addition to heavy periods and severe pain, this condition causes infertility miscarriage, and complications during pregnancy and childbirth, such as preterm labour, and placental problems, which can be life-threatening to the mother and her unborn baby.
Despite this being a common condition, there isn't much research looking into it, so we know very little about it and what treatments options are best. There have been some recent advances in imaging techniques allowing more women to be diagnosed with adenomyosis with just an ultrasound or MRI scan. Unfortunately, there are still no specific treatments available for these women.
The majority of the limited available treatments are not suitable for women who wish to have children. These treatments include pain relief, hormonal medications, or an operation to remove the womb (a hysterectomy). Therefore, there is a pressing need to find new treatments for adenomyosis to enable women to have effective symptom relief without impacting on their fertility, or resorting to major surgery.
The aim of my research is to improve the understanding of adenomyosis at a molecular level with a view to finding potential new treatments. I plan to ask at least 10 women who are having a hysterectomy for adenomyosis if they want to take part in this research. This will involve collecting tissue samples (biopsies) from the womb after it has been removed, so the women will not be subjected to any additional risk or discomfort.
After collecting the biopsies, I will use a fine laser and a microscope to cut out the cells in the normal endometrium and those from adenomyosis buried in the myometrium. I will then compare the genes found in the normal endometrium with the adenomyosis, to identify any key differences in these diseased cells. Genes carry the instructions that cells use to make proteins and perform certain functions. I will also perform experiments to compare the proteins produced by the normal endometrial cells with the adenomyotic cells. In order to identify proteins, I will use antibodies (binding molecules) which are linked to a fluorescent dye or enzyme. Once the protein binds with the antibody, it can be seen under a microscope. This will identify specific markers in adenomyosis which could be targeted by new treatments.
Moreover, I will produce a three-dimensional (3D) model of adenomyosis, to improve our understanding of how the adenomyosis grows within the myometrium, using advanced technology to scan a biopsy from women with adenomyosis, and reconstruct the images virtually. This may identify new modes to deliver treatment for adenomyosis, for example, using an abundant blood supply to deliver treatment if seen.
Finally, in the lab, I will grow the cells from the uterine biopsies to mimic a 3D adenomyosis lesion. This will serve as a laboratory model where new treatments for adenomyosis can be tested safety for efficacy.
This fundamental research will help us understand what causes adenomyosis, with a view to developing novel treatments for women suffering with this condition.
Patient and public involvement was sought and this lay summary was reviewed by patients with heavy menstrual bleeding.

The aim of this project is to understand the pathogenesis of adenomyosis, identify therapeutic targets, and facilitate the development and testing of novel, fertility sparing treatments. There are 3 objectives:
1. To determine the transcriptional differences of cells within adenomyotic lesions compared with the specific anatomical regions of the correctly located (eutopic) endometrium.
2. To identify a specific panel of markers that characterise the epithelium and stroma of adenomyotic lesions, and determine their 3D microarchitecture.
3. To develop a novel in-vitro 3D culture model of adenomyosis.
LCM will be used to isolate epithelial and stromal cells from the endometrium and the adenomyotic lesions, from 10 full thickness uterine wall biopsies obtained at hysterectomy from women with adenomyosis. RNA sequencing of endometrial cells from the aforesaid locations will reveal their key transcriptional differences, and potential surface markers specific to adenomyosis. The activity of functional pathways relevant to the candidate genes will be ascertained by immunostaining in full thickness uterine wall biopsies (n=10), and Hyperion IMC (n=3). A 3D reconstruction of adenomyosis will determine its microarchitecture and surrounding structures, using light sheet fluorescence microscopy and digital scanning of consecutive H&E stained endometrial biopsy sections from women with adenomyosis. Finally, a novel in-vitro 3D culture model of adenomyosis will be produced, for pre-clinical drug testing, using primary epithelial organoid cultures from women with adenomyosis (n=10), seeded into a collagen hydrogel containing endometrial stromal cells, and co-culturing these with a primary myometrial cells. The outcomes will inform on the key transcriptomic and phenotypic signature of cells within adenomyotic lesions, which could be targeted with novel therapeutic approaches such as nanotechnology, and can be tested on the 3D in-vitro cell culture model in the future.