Endometriosis is a chronic, estrogen-dependent disorder where inflammation contributes to disease-associated symptoms of pelvic pain and infertility. Immune dysfunction includes insufficient immune lesion clearance, a pro-inflammatory endometrial environment, and systemic inflammation. Comprehensive understanding of endometriosis immune pathophysiology in different hormonal milieu and disease severity has been hampered by limited direct characterization of immune populations in endometrium, blood, and lesions. Simultaneous deep phenotyping at single cell resolution of complex tissues has transformed our understanding of the immune system and its role in many diseases. Herein, we report mass cytometry and high dimensional analyses to study immune cell phenotypes, abundance, activation states, and functions in endometrium and blood of women with and without endometriosis in different cycle phases and disease stages. A case-control study was designed. Endometrial biopsies and blood (n=60) were obtained from women with (n=20/n=17, respectively) and without (n=14/n=9) endometriosis in the proliferative and secretory cycle phases. Two mass cytometry panels were designed; one broad panel and one specific for mononuclear phagocytic cells (MPC), and all samples were multiplexed to characterize both endometrium and blood immune composition at unprecedented resolution. We combined supervised and unsupervised analyses to finely define the immune cell subsets with an emphasis on MPC. Then, association between cell types, protein expression, disease status, and cycle phase were performed. The broad panel highlighted a significant modification of MPC in endometriosis; thus, they were studied in detail with an MPC-focused panel. Endometrial CD91⁺ macrophages overexpressed SIRPα (phagocytosis inhibitor) and CD64 (associated with inflammation) in endometriosis, and they were more abundant in mild versus severe disease. In blood, classical and intermediate monocytes were less abundant in endometriosis, whereas plasmacytoid dendritic cells and non-classical monocytes were more abundant. Non-classical monocytes were higher in severe versus mild disease. A greater inflammatory phenotype and decreased phagocytic capacity of endometrial macrophages in endometriosis are consistent with defective clearance of endometrial cells shed during menses and in tissue homeostasis, with implications in endometriosis pathogenesis and pathophysiology. Different proportions of monocytes and plasmacytoid dendritic cells in blood from endometriosis suggest systemically aberrant functionality of the myeloid system opening new venues for the study of biomarkers and therapies for endometriosis.

A total of 34 fresh endometrial tissues (Control = 14, Endometriosis = 20) and 26 blood samples (Control = 9, Endometriosis = 17) were collected from women with and without endometriosis undergoing surgery (total n = 60). Diagnosis was made by the physician and pathologists from UCSF and stage of the disease was determined following the ASRM classification system. Controls were determined as women not suffering from endometriosis. Adenomyosis cases were also excluded as the two diseases share many features. However, some patients did show presence of myomas or polyps. Nonetheless, all controls were endometriosis-free, or the disease was never diagnosed. Cycle phase was determined by following Noyes et al. system. Clinical features of the patients were collected only by authorized personnel by using REDCap. Only patients in reproductive age were included in the study. In addition, all patients presenting immune-related comorbidities, such as lupus, endometritis, or any type of immune diseases, were excluded from the study, as well as patients positive for HIV, HVB and HVC. Moreover, patients were not exposed to hormone therapies for at least 3 months prior to collection. All samples were obtained between 2019 and 2021 under the auspices of the University of California, San Francisco (UCSF) Institutional Review Board Procotol #: IRB#10-03964, using standard operating and sampling procedures.

Endometrial samples: Endometrium was obtained either by endometrial biopsy using a Pipelle catheter or from hysterectomy specimens. Tissues were placed into transport medium and processed within 5 hours following collection where they were first washed with serum containing media (SCM) and then digested mechanically and enzymatically using a mix of collagenase IV and hyaluronidase. After one hour of digestion at 37°C under rotation, for live/dead discrimination, samples were processed for incorporation of cisplatin, and fixed for further usage. Briefly, the single cell suspension was washed with FACS/EDTA buffer (PBS supplemented with 2% FBS and 2mM EDTA). Cells were counted and an appropriate amount of cisplatin (25mM per 1-6 million cells) was added to the suspension (4ml PBS/EDTA per 1-6 million cells) for exactly 60 seconds at room temperature. Then the cells were quenched with CyFACS (metal contaminant-free PBS supplemented with 0.1% BSA and 0.1% sodium azide). Finally, cells were fixed with 1.6% formaldehyde for 10 minutes, washed three times in CyFACS, and stored at -80°C until further use.

Peripheral blood mononuclear cells (PBMCs): Blood was collected in collection tubes containing anticoagulant acid citrate dextrose (ADC) Solution B. Ficoll was slowly added to the bottom of the blood in a falcon tube at a ratio of 2:1. The samples were then centrifuged at 2000 rpm for 30 minutes at RT. After centrifugation, the supernatant was removed and the PBMCs layer was carefully collected. PBMCs were washed twice with FACS buffer, and the number of cells was then counted. The same protocol above was used to incorporate cisplatin, fix, and store the cells.

Data processing: The fcs. files obtained from the instrument were concatenated, normalized to EQ^TM calibration beads and de-barcoded using CyTOF software (Fluidigm). This study is comprised of eight different sample groups: control (Ctrl) and endometriosis (Endo) eutopic endometrium (EM) in the proliferative (PE) and secretory (SE) phases (Ctrl_EM_PE, Ctrl_EM_SE, Endo_EM_PE, Endo_EM_SE) and control and blood (PBMCs) in the proliferative and secretory phases (Ctrl_PBMC_PE, Ctrl_PBMC_SE, Endo_PBMC_PE, Endo_PBMC_SE). Normalized data from the broad panel were imported to FlowJo to perform manual gating and we performed unsupervised analysis of the manually gated CD45⁺ cells. Then, unsupervised analysis of the manually-gated myeloid cells of interest (CD45+, CD3-, CD56-, CD66b-)(including macrophages, monocytes, dendritic cells, and plasmacytoid dendritic cells) was also performed. 

This dataset contains two types of datasets. One for the broad panel (n=17, all endometrial samples) and one for the focused panel (n=39, n=31 for endometrium and n=26 for PBMC). We have included here a metadata excel file for each panel. In addition, we have also included the two CyTOF panels used in this study, which indicate the channels and markers for each channel. For the brad panel, the fcs. files contain CD45+ manually gated live cells. For the focused panel, cells were manually gated obtaining myeloid cells (and a cluster of putative B cells). The files contain cells that are live CD45+, CD3-, CD66b-, CD56-, and CD56-.