Zou et al. Cell Biosci (2021) 11:98 https://doi.org/10.1186/s13578-021-00613-5 Cell & Bioscience RESEARCH Open Access Cell subtypes and immune dysfunction in peritoneal fuid of endometriosis revealed by single-cell RNA-sequencing Gen Zou†, Jianzhang Wang†, Xinxin Xu†, Ping Xu, Libo Zhu, Qin Yu, Yangying Peng, Xinyue Guo, Tiantian Li and Xinmei Zhang* Abstract Background: Endometriosis is a refractory and recurrent disease and it afects nearly 10% of reproductive-aged women and 40% of infertile patients. The commonly accepted theory for endometriosis is retrograde menstruation where endometrial tissues invade into peritoneal cavity and fail to be cleared due to immune dysfunction. Therefore, the comprehensive understanding of immunologic microenvironment of peritoneal cavity deserves further investiga- tion for the previous studies mainly focus on one or several immune cells. Results: High-quality transcriptomes were from peritoneal fuid samples of patients with endometriosis and control, and frstly subjected to 10 genomics single-cell RNA-sequencing. We acquired the single-cell transcriptomes of 10,280 cells from endometriosis× sample and 7250 cells from control sample with an average of approximately 63,000 reads per cell. A comprehensive map of overall cells in peritoneal fuid was frst exhibited. We unveiled the hetero- geneity of immune cells and discovered new cell subtypes including T cell receptor positive (TCR ) macrophages, proliferating macrophages and natural killer dendritic cells in peritoneal fuid, which was further verifed+ by double immunofuorescence staining and fow cytometry. Pseudo-time analysis showed that the response of macrophages to the menstrual debris might follow the certain diferentiation trajectory after endometrial tissues invaded into the peritoneal cavity, that is, from antigen presentation to pro-infammation, then to chemotaxis and phagocytosis. Our analyses also mirrored the dysfunctions of immune cells including decreased phagocytosis and cytotoxic activity and elevated pro-infammatory and chemotactic efects in endometriosis. Conclusion: TCR macrophages, proliferating macrophages and natural killer dendritic cells are frstly reported in human peritoneal+ fuid. Our results also revealed that immune dysfunction happens in peritoneal fuid of endome- triosis, which may be responsible for the residues of invaded menstrual debris. It provided a large-scale and high- dimensional characterization of peritoneal microenvironment and ofered a useful resource for future development of immunotherapy. Keywords: Endometriosis, Single-cell RNA-sequencing, Peritoneal fuid, Cell profling, Immune dysfunction Background Endometriosis is a chronic infammatory disease and *Correspondence: [email protected] afects nearly 10% of reproductive-aged women and as †Gen Zou, Jianzhang Wang and Xinxin Xu contributed equally to this work high as 40% of infertile women [1]. It is clinically mani- Department of Obstetrics and Gynecology, Women’s Hospital, School fested with severe pelvic pain and reduced fertility and of Medicine, Zhejiang University, 1 Xue Shi Road, Hangzhou 310006, is characterized by the presence and growth of endome- Zhejiang, People’s Republic of China trial tissue outside the uterus, which seriously reduces © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdo- main/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zou et al. Cell Biosci (2021) 11:98 Page 2 of 17 the life quality of patients and causes a heavy burden on the pathogenesis of endometriosis, it warrants an unbi- the healthcare [2, 3]. Te etiology is still incompletely ased characterization of cell contents and scRNA-seq of understood and “retrograde menstruation” is one widely all the cells in peritoneal fuid. Here, we found that the accepted theory that the refux accounts for the accu- peritoneal microenvironment is mainly composed of dif- mulation of menstrual debris in peritoneal cavity [4, 5]. ferent immune cells. We further found that the immune However, retrograde menstruation occurs in almost all cells in endometriosis were dysfunctional with decreased cycling women, while only a minority of them develops phagocytosis and cytotoxic activity and elevated pro- endometriosis, implying that additional factors contrib- infammatory and chemotactic efects. Importantly, our ute to the development of endometriosis [6]. fndings ofer a useful resource for understanding pathol- Immune cells contribute to scavenging menstrual ogy of endometriosis and potential immunotherapy of debris in cycling women [7]. One of the possible causes endometriosis. of endometriosis is the defective immune response to the refuxed menstrual debris in peritoneal cavity, which determines the survival and implantation of ectopic Methods endometrial cells and lesion formation [8]. Accumulated Ethics and sample collection evidence over the past decade has suggested that devel- Tis project was approved by the Ethics Committee of opment of endometriosis is accompanied with sustained Women’s Hospital, School of Medicine, Zhejiang Univer- peritoneal infammation, including altered immune cell sity (IRB-20200003-R). Included patients supplied writ- contents in peritoneal fuid and ectopic lesions, as well ten informed consent for collection of specimens and as changed immune cells cytotoxicity and activation [9]. analyses of the derived genetic materials prior to their Alterations in both innate and adaptive immunity con- participation. tribute to the pathogenesis of endometriosis [9–11]. As Peritoneal fuid samples were collected from 39 endo- the front line of innate immunity, macrophages com- metriosis patients and 27 non-endometriosis controls prise the largest immune cell population in peritoneal undergoing surgery (details in Additional fle 2: Table S1). fuid of both healthy women and endometriosis patients All included patients did not receive hormone treatment [12]. Tey are complex cells at the center of this elusive in the 6 months and all samples were collected in prolif- condition, which are critical for the growth, vasculariza- erative phase. Among them, cell suspensions from one tion and innervation of endometriosis lesions. Previous endometriosis patient and one control patient with sep- studies have demonstrated functional disorders of mac- tate uterus were subjected to scRNA-seq and the other rophage in endometriosis. However, it is still controver- 64 samples were applied for the validation using double sial whether macrophages in peritoneal fuid exhibit a immunofuorescence or fow cytometry. Samples were pro-infammatory or pro-repair phenotype [13]. Recent collected during laparoscopic surgery before any surgical studies have revealed that they are complex and hetero- procedure to avoid contamination from blood. Samples geneous in the pathology of endometriosis [14]. In addi- were transported to the laboratory in a cold chain within tion to macrophages, other immune cells also have been 30 min and used for subsequent experiments. proposed to play important roles in the pathogenesis of endometriosis. Decreased cytotoxicity of natural killer (NK) cells had been reported in peritoneal fuid of endo- Cell preparation metriosis [15]. An increased proportion of regulatory T Cells were pelleted from peritoneal fuid and washed (Treg) cells in peritoneal fuid of women with endometri- three times. Red blood cells were lysed using Ammo- osis has been reported [16]. Dendritic cells (DCs), mast nium-Chloride-Potassium Lysing Bufer (Gibco, USA) cells and B cells have been observed to be changed as well according to the manufacturer’s instructions. Samples [17–19]. were next diluted with PBS containing 0.04% Bovine Previous studies mainly focus on one or several Serum Albumin (Sigma, USA) to the density of about immune cells and are lack of comprehensive investiga- 6 1 × ­10 cells/mL. 10 µL of this cell suspension was mixed tion, which leads to a failure to discover heterogeneous with 10 μL 0.4% trypan blue solution (Sigma, USA) and cell contents at an unbiased scale. Recent advance in sin- counted using an automated cell counter (Bio-rad, USA) gle-cell RNA-sequencing (scRNA-seq) has the potential to determine the density of live cells. Cell viability of sam- to resolve heterogeneous cell populations at an unprec- ples used for single cell sequencing was 94% for endo- edented scale [20]. It has been used to discriminate cell metriosis sample and 86% for control sample. Cells were types in healthy tissues or tumors, to explore immune cell maintained on ice whenever possible throughout the dis- heterogeneity and to reveal