Defining chronic endometritis using emerging transcriptomic and proteomic technologies to improve its diagnosis

Abstract Unexplained infertility accounts for up to one third of infertility cases and among these, chronic endometritis (or CE) affects up to 40%, including those with recurrent implantation failure (RIF). CE is defined as chronic endometrial inflammation, often of microbial origin. However, some cases of CE are due to non-infectious or unknown factors. Patients typically present with non-specific or no symptoms making CE difficult to diagnosis. CE diagnosis is mainly based on CD138+ plasma cells in the stromal region of an endometrial biopsy - with no consensus on the number or fraction of plasma cells needed. Infertile patients diagnosed with CE are treated with antibiotics, with up to 60-80% success. Thus, many CE patients ‘fail’ antibiotic therapy. Without strict diagnostic criteria and the inability to distinguish infectious vs. non-infectious CE, the field is ripe for comprehensively profiling the endometrium in CE to improve diagnosis. We hypothesize that emerging transcriptomic and proteomic approaches applied to endometrial biopsies will define cell-specific phenotypic and gene expression alterations in immune cell and non-immune cell populations in the endometrium using paired (within person) CE biopsies pre- and post-antibiotic treatment and biopsies from healthy controls. Additionally, we hypothesize that non-invasively collected menstrual effluent (ME) can serve as a non-invasive source of endometrial tissue to study and diagnose CE. We will apply both transcriptomic and proteomic methods developed by our lab for studying the endometrium in the setting of endometriosis to better define CE in patients with unexplained infertility and RIF. In aim 1 we will employ single cell RNA-sequencing (scRNA- Seq, transcriptomic) and multiplexed Cell DIVE imaging (proteomic) to analyze endometrial biopsies from infertile patients with RIF and suspected CE. CE-positive cases receive antibiotics and are then re-biopsied to confirm CE-negative status before future IVF cycles (providing paired CE-pos-CE-neg or CE-pos-CE-pos biopsies from the same patients). Additionally, we will compare biopsy assessments from CE-positive and CE- negative cases to ‘healthy control’ biopsies. In aim 2 we will apply our well-developed approaches to the analysis of paired menstrual effluent (ME) tissues from the same cohort (CE-neg vs. CE-pos cases and vs. healthy controls) as in Aim 1 and test for dysfunctional stromal cells. Based on our prior studies applying scRNA-Seq and Cell DIVE to ME/endometrial tissue in the setting of endometriosis, we expect to reveal CE- associated molecular and cellular phenotypic changes that inform disease-associated pathogenic alterations underlying CE. Finally, we will determine the suitability of ME for non-invasively studying and diagnosing CE. Results from these proposed studies are expected to expand investigative approaches to studying CE, defining CE phenotypes (e.g. infectious vs. non-infectious), and improving CE diagnosis.