Biomarkers for Endometriosis

Background:

Endometriosis is a benign inflammatory disease defined by the presence of endometrial glands and stroma in areas outside the uterus[1]. It is estimated to affect 5%-15% of women of reproductive age [2] and is even more prevalent among women with chronic pelvic pain and infertility [3]. Endometriotic implants may be located anywhere within the pelvis, including the ovaries, pelvic peritoneum, rectovaginal septum, uterosacral ligaments, and cul-de sac. The pathophysiology of endometriosis still remains unclear. The etiologic mechanism most widely accepted is retrograde menstruation resulting in tubal backflow of endometrial cells into the pelvis during menses [4]. Over time, these cells may adhere to peritoneal surfaces, proliferate, and progressively grow. The amount, duration, and frequency of pelvic exposure to menstrual bleeding may also determine the severity of endometriosis [5]. At present, the diagnosis of endometriosis can only be definitively made by laparoscopy and biopsy of endometrial lesions for histologic confirmation. Since these are invasive procedures and usually performed at the late stages, there is an urgent need to identify non-invasive biomarkers to effectively indicate endometriosis especially at the early stages for better clinical management.

Rational:

Many genes and proteins have been proposed as biomarkers for diagnosis and follow up of endometriosis [6, 7], but none used at the present time is sensitive and specific enough either to diagnose endometriosis nor to determine the effectiveness of treatment of endometriosis. Synuclein gamma (SNCG), a member of the synuclein family of neuronal proteins, is involved in cellular proliferation by interacting with the mitotic checkpoint kinase BubR1, and inhibiting its regulatory role on cellular proliferation [8]. During the last decade, several studies have demonstrated that SNCG is correlated with poor outcome of breast cancer, ovarian cancer, colon cancer and pancreatic cancer. Interestingly, SNCG has previously been shown to have elevated expression ranging from 5- to 53-fold in ovarian endothelial cells of endometriosis lesions compared with eutopic endometrium from the same individual [9]. Similarly, B-Cell Lymphoma 6 (BCL6) protein is encoded by the BCL6 oncogene and is a zinc finger transcription factor that regulates the transcription of signal transducer and activators of transcription (STAT)-dependent IL-4 responses of B-cells [10]. Previously, BCL6 was shown to express in the endometrial epithelial cells and B-cells in lymph node germinal centres [11,12]. In addition, over-expression of BCL6 has been reported in endometrial biopsies and has been linked to the progesterone resistance and pathogenesis of endometriosis [13]. Together, both of these proteins (SNCG and BCL6) have been linked to endometriosis. However, their diagnostic/prognostic utility by non-invasive means such as measurement in peripheral blood and menstrual fluid of patients with endometriosis remains to be investigated.

Hypothesis:

We hypothesize that SNCG and BCL6 concentrations are altered in patients with endometriosis and measurements of circulating SNCG and BCL6 levels in serum/menstrual blood could be used for diagnosis and/or prognosis of endometriosis.

OBJECTIVES

Objectives:

The aim of this study is to measure the SNCG and BCL6 concentrations in peripheral and menstrual fluid from endometriosis patients compared to age-matched healthy subjects or in patients after the treatment of endometriosis. We have the following specific objectives:

• Identification of endometriosis and control groups and sample collection: Eligible patients with active endometriosis (n=100) confirmed through standard clinical diagnosis will be recruited along with age matched healthy control subjects (n=50). Peripheral blood and menstrual fluid samples will be collected from both of these groups. In addition, the endometriosis group will be followed post treatment (either medically or surgically) and their blood and menstrual fluids samples will be collected at the 3rd and 6th month safter treatment. These collected samples will be processed for serum/supernatant isolation and stored at -80 C until further analysis.
• Analysis of circulatory SNCG and BCL6 levels using ELISA method: SNCG and BCL6 levels in serum/menstrual supernatant will be quantitated using specific ELISA kits and will be compared for significant differences in patients with endometriosis from control subjects as well as their changes before and after the treatment in endometriosis group.

Expected Outcome and significance:

We expect that SNCG and BCL6 concentrations in patients with endometriosis would be significantly higher compared to age-matched healthy subjects. In addition, we expect that SNCG and BCL6 concentrations may also show differences in samples of endometriosis patients treated with suppression medication or after surgery. This study aims to validate their diagnostic/prognostic potential of SNCG and BCL6 concentrations as non-invasive biomarkers for endometriosis.

STUDY DESIGN This study will focus on measuring circulatory SNCG and BCL6 concentrations in patients with endometriosis compared to age-matched healthy subjects without endometriosis. Patients with confirmed endometriosis, and the healthy control group will be recruited at TRIO Fertility following informed consent. Peripheral blood samples and menstrual samples will be collected from both groups and serum/supernatant from these samples will be used for measuring the baseline differences of SNCG and BCL6 levels. In addition, post-treatment changes in these proteins will also be measured in samples collected from endometriosis patients at the 3rd and 6th month following endometriosis suppression with dienogest, Lupron depot and addback, or Myfembree, or after laparoscopic surgery with excision or vaporization of endometriosis lesions. Analysis of circulatory SNCG/BCL6 levels will be performed using SNCG and BCL6 specific enzyme linked immunosorbent assays (ELISA), respectively, and will be compared for significant differences between the endometriosis and the control group, and pre and post treatment in endometriosis group.

Inclusion criteria

for Endometriosis Group:

• Women of reproductive age group (18Y-45Y).
• Women with active endometriosis diagnosed by ultrasound/MRI/laparoscopy.

Exclusion criteria

for Endometriosis Group:

• Women <18Y and >45Y.
• Women with endometriosis during stimulation with gonadotropins.
• Current or prior history of uterine, ovarian, colon, pancreatic, breast or any other type of cancer.
• Any prior history of chronic illness.
• Pregnant women.

Inclusion criteria

for Control Group:

• Women of reproductive age group (18Y-45Y).
• Women without endometriosis and any other gynaecological problem.

Exclusion criteria

for Endometriosis Group:

• Women <18Y and >45Y
• Prior history of any disease including cancer or chronic illness.
• Pregnant women.