International Journal of Molecular Sciences Article Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium Júlia Vallvé-Juanico 1,2 , Cristian Barón 2 , Elena Suárez-Salvador 3 , Josep Castellví 4 , Agustín Ballesteros 1 , Antonio Gil-Moreno 2,3,5 and Xavier Santamaria 1,2,6, * 1 Department of Reproductive Medicina, IVIRMA-Barcelona, 08017 Barcelona, Spain; julia.vallve@vhir.org (J.V.-J.); agustin.ballesteros@ivirma.com (A.B.) 2 Group of Biomedical Research in Gynecology, Vall Hebron Research Institute (VHIR) and University Hospital, 08035 Barcelona, Spain; crbaronv@gmail.com (C.B.); antonioimma@yahoo.es (A.G.-M.) 3 Department of Gynecology, Vall d’Hebron University Hospital, 08035 Barcelona, Spain; elenafeb@yahoo.es 4 Department of Pathology, Vall d’Hebron University Hospital, 08035 Barcelona, Spain; joscastellvi@vhebron.net 5 Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain 6 Igenomix Foundation, Paterna, 46980 Valencia, Spain * Correspondence: Xavier.santamaria@ivirma.com; Tel.: +34-932-06-30-00  Received: 8 November 2018; Accepted: 15 December 2018; Published: 21 December 2018  Abstract: Endometriosis is characterized by the abnormal presence of endometrium outside of the uterus, resulting in pelvic pain and infertility. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has been postulated to be a marker of stem cells in the endometrium. However, LGR5+ cells have a macrophage-like phenotype in this tissue, so it is unclear what role LGR5+ cells actually play in the endometrium. Macrophages serve an important function in the endometrium to maintain fertility, while LGR5+ cells generally have a role in tumor progression and are involved in invasion in some cancers. We sought to determine whether LGR5+ cells vary across the menstrual cycle in women with endometriosis and whether there are implications for LGR5 in the aggressiveness of endometriosis and reproductive outcomes. We performed immunofluorescence, flow cytometry, and primary culture in vitro experiments on eutopic and ectopic endometrium from healthy and endometriosis patients and observed that neither LGR5+ cells nor LGR5 expression varied throughout the cycle. Interestingly, we observed that LGR5+ cell percentage overexpressing CD163 (anti-inflammatory marker) was higher in healthy endometrium, suggesting that in endometriosis, endometrium presents a more pro-inflammatory phenotype that likely leads to poor obstetric outcomes. We also observed higher levels of LGR5+ cells in ectopic lesions compared to eutopic endometrium and specifically in deep infiltrating endometriosis, indicating that LGR5 could be involved in progression and aggressiveness of the disease. Keywords: LGR5; endometrium; endometriosis; menstrual cycle; macrophages 1. Introduction Endometriosis is a chronic estrogen-dependent disease characterized by the presence of endometrial tissue outside the uterine cavity. Primary symptoms of the disease, which affects approximately 10% of reproductive age women, are acute pelvic pain and/or infertility/subfertility [1]. Endometrium, which is composed of stromal and epithelial compartments, contains many immune cells and is a very dynamic tissue that is tightly regulated by ovarian hormones. The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) is a seven transmembrane receptor described as a stem cell marker in a variety of tissues, including the small intestine and hair Int. J. Mol. Sci. 2019, 20, 22; doi:10.3390/ijms20010022 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 22 2 of 12 follicles [2–4]. LGR5 has been identified in endometrium [5,6], but its role in endometrial function is unclear. Recently, it was discovered that LGR5-positive cells (LGR5+ ) from healthy endometrium have a hematopoietic origin [7]. Approximately half of the population of LGR5+ cells found in the endometrium co-express CD45, a leukocyte marker, and CD163, a monocyte and macrophage specific marker, suggesting a myeloid nature to the cells. This expression pattern was also observed by our group in LGR5+ cells from eutopic endometrium from women with endometriosis [8]. Interestingly, LGR5+ cells seem to remain constant throughout the menstrual cycle in normal human eutopic endometrium [7,9], although it was recently described that LGR5 expression decreases during the secretory phase [6]. We previously described a special subset of LGR5+ cells that express unique genes in the eutopic endometrium of women with deep infiltrating endometriosis (DIE) compared to other types of endometriosis. These genes are related to immune system responses, hematological system development, and infertility [8]. Thus, we believe that LGR5+ cells could be implicated in aggressiveness and reproductive outcomes of the disease based on the function of these co-expressed genes. LGR5+ cells have a macrophage-like phenotype, and macrophages have been shown to increase in the secretory and menstrual phases of the menstrual cycle in normal eutopic endometrium [10]. However, this increase has not been observed in eutopic endometrium of women with endometriosis [11]. Macrophages have a role in embryo implantation failure and are involved in poor reproductive outcomes [12]. Moreover, this immune cell population is increased in the peritoneal fluid and endometriotic lesions of women with endometriosis [13]. However, variation of these types of cells throughout the menstrual cycle in women with endometriosis has not been investigated. We assessed whether LGR5+ cells vary throughout the menstrual cycle, along with the percentage of LGR5+ cells in ectopic versus eutopic endometrium, as these cells may have implications for reproductive outcomes in endometriosis. 2. Results 2.1. LGR5 Does Not Vary Throughout the Menstrual Cycle in Women with Endometriosis To determine whether LGR5 varies throughout the menstrual cycle, we used three experimental approaches: immunofluorescence, in vitro assay, and flow cytometry. We used endometrial biopsies and ectopic lesions in order to answer this question. The characteristics of the patients are shown in Table 1. Table 1. Patients used in the study. Type of Eutopic Mached Ectopic Technique Patient ID Type of Patient Endometriosis Endometrium Endometrium Used 1 Control - yes - FC 2 Control - yes - FC 3 Control - yes - FC 4 Control - yes - FC 5 Control - yes - FC 6 Control - yes - FC 7 Control - yes - FC 8 Control - yes - FC 9 Control - yes - FC/CC 10 Control - yes - FC/CC 11 Control - yes - FC/CC 12 Control - yes - FC/CC 13 Control - yes - IF 14 Control - yes - IF 15 Control - yes - IF 16 Control - yes - IF Int. J. Mol. Sci. 2019, 20, 22 3 of 12 Table 1. Cont. Type of Eutopic Mached Ectopic Technique Patient ID Type of Patient Endometriosis Endometrium Endometrium Used 17 Control - yes - IF 18 Control - yes - IF 19 Control - yes - IF 20 Control - yes - IF 21 Control - yes - IF 22 Control - yes - IF 23 Control - yes - IF 24 Control - yes - IF 25 Control - yes - IF 26 Control - yes - IF 27 Control - yes - IF 28 Control - yes - IF 29 Control - yes - IF 30 Control - yes - IF 31 Control - yes - IF 32 Control - yes - IF 33 Control - yes - IF 34 Control - yes - IF 35 Control - yes - IF 36 Control - yes - IF 37 Endometriosis Ovarian yes - FC 38 Endometriosis Ovarian yes - FC 39 Endometriosis Ovarian yes - FC 40 Endometriosis Ovarian yes - FC 41 Endometriosis Ovarian yes yes FC 42 Endometriosis Ovarian yes yes FC 43 Endometriosis Ovarian yes yes FC 44 Endometriosis Ovarian yes yes FC 45 Endometriosis DIE yes yes FC 46 Endometriosis DIE yes yes FC 47 Endometriosis DIE yes yes FC 48 Endometriosis DIE yes yes FC 49 Endometriosis DIE yes yes FC 50 Endometriosis DIE yes yes FC 51 Endometriosis DIE yes - FC 52 Endometriosis DIE yes - FC 53 Endometriosis DIE yes - FC 54 Endometriosis DIE yes - FC 55 Endometriosis Pelvic yes yes FC 56 Endometriosis Pelvic yes - FC 57 Endometriosis Pelvic yes - FC 58 Endometriosis Adenomyosis yes - FC 59 Endometriosis Adenomyosis yes yes FC 60 Endometriosis Adenomyosis yes - FC 61 Endometriosis Adenomyosis yes yes FC 62 Endometriosis DIE yes - CC 63 Endometriosis DIE yes - CC 64 Endometriosis DIE yes - CC 65 Endometriosis ND yes - IF 66 Endometriosis ND yes - IF 67 Endometriosis ND yes - IF 68 Endometriosis ND yes - IF 69 Endometriosis ND yes - IF 70 Endometriosis ND yes - IF 71 Endometriosis ND yes - IF 72 Endometriosis ND yes - IF 73 Endometriosis ND yes - IF 74 Endometriosis ND yes - IF Int. J. Mol. Sci. 2019, 20, 22 4 of 12 Table 1. Cont. Type of Eutopic Mached Ectopic Technique Patient ID Type of Patient Endometriosis Endometrium Endometrium Used 75 Endometriosis ND yes - IF 76 Endometriosis ND yes - IF 77 Endometriosis ND yes - IF 78 Endometriosis ND yes - IF 79 Endometriosis ND yes - IF 80 Endometriosis ND yes - IF 81 Endometriosis ND yes - IF 82 Endometriosis ND yes - IF 83 Endometriosis ND yes - IF 84 Endometriosis ND yes - IF 85 Endometriosis ND yes - IF 86 Endometriosis ND yes - IF 87 Endometriosis ND yes - IF 88 Endometriosis ND yes - IF FC: flow cytometry; CC: cell culture; IF: immunofluorescence; ND: non-determined. 2.1.1. Immunofluorescence Analysis Calculations of fluorescence intensity mean (FIM) for LGR5 throughout the menstrual cycle showed no significant differences between epithelial and stromal compartments in control endometrium and no significant differences in the case of endometriosis (Figure 1A). Additionally, there were no significant differences between phases of the menstrual cycle in either group (Figure 1B). However, when comparing LGR5 expression in the eutopic endometrium from healthy patients and endometriosis patients, we observed a statistically significant increase in LGR5 expression in proliferative, secretory, and menstrual phases in healthy women (Figure 1C). An example of the immunofluorescence in eutopic endometrium is shown in Figure 1D. Figure 1. Immunofluorescence measurement of LGR5 expression throughout the menstrual cycle in eutopic endometrium. (A) Epithelial and stromal expression of LGR5 in control and endometriosis tissue in five phases of the menstrual cycle. (B) Total tissue expression across five phases of the menstrual cycle in control and endometriosis tissues. (C) Differences in LGR5 expression throughout the menstrual cycle between control and endometriosis tissue groups (t-test for each phase; proliferative: p = 0.0242; secretory: p = 0.0424; menstruation: p = 0.0121). (Control: n = 24; endometriosis: n = 24). (D) Example of immunofluorescence of LGR5 in eutopic endometrium (early secretory phase). In blue: DAPI; in red: LGR5. * p < 0.05. Int. J. Mol. Sci. 2019, 20, 22 5 of 12 Int. J. Mol. Sci. 2018, 19, x 5 of 13 2.1.2. In vitro Studies 2.1.2. In vitro Studies In vitro experiments using endometrial stromal fibroblasts (eSF) cells treated with estradiol (E ) In vitro experiments using endometrial stromal fibroblasts (eSF) cells treated with estradiol (E22) for six days, and with E2 and progesterone (P4 ) for six more days, showed that primary culture for six days, and with E2 and progesterone (P4) for six more days, showed that primary culture cells cells mimicked proliferative and secretory phases of the menstrual cycle. Although non-significant, mimicked proliferative and secretory phases of the menstrual cycle. Although non-significant, a a tendency in increase in expression of CYR61, which increases during the proliferative phase, tendency in increase in expression of CYR61, which increases during the proliferative phase, was was observed and a tendency in increase of DKK1, which increases during the secretory phase, observed and a tendency in increase of DKK1, which increases during the secretory phase, was also was also observed at days 6 and 12, respectively, in both groups when compared to the untreated observed at days 6 and 12, respectively, in both groups when compared to the untreated control control (Figure 2A). No differences in cell morphology were observed. In addition, no significant (Figure 2A). No differences in cell morphology were observed. In addition, no significant variation variation in LGR5 throughout the menstrual cycle was observed in control or endometriosis samples. in LGR5 throughout the menstrual cycle was observed in control or endometriosis samples. Interestingly, a non-significant decrease in LGR5 was observed in the proliferative phase of the Interestingly, a non-significant decrease in LGR5 was observed in the proliferative phase of the treatment group in control samples, while we observed a non-significant increase in the secretory treatment group in control samples, while we observed a non-significant increase in the secretory phase of endometriosis samples (Figure 2B). phase of endometriosis samples (Figure 2B). Figure Figure 2. 2. In In vitro vitro analysis analysis ofof LGR5 LGR5 expression expression throughout throughout the the menstrual menstrual cycle cycle in in endometrial endometrial stromal stromal fibroblast primaryculture fibroblast primary cultureafter aftertreatment treatment (E2(Eand 2 andE2 PE42Pfor 4 for sixsix andand six six more more days, days, respectively). respectively). (A) (A) Left Left panels panels show show expression expression of CYR61 of CYR61 (marker (marker of of thethe proliferativephase) proliferative phase)andandDKK1 DKK1(marker (marker of of the the secretory secretory phase) phase) in inthe thecontrol controlgroup group(n(n= = 4);4); right panels right panels showshowexpression expressionof CYR61 of CYR61andand DKK1DKK1 in the in endometriosis the endometriosis group (n =(n3). group (B)(B) = 3). Upper Upper panel panel shows showsexpression expressionofofLGR5LGR5inin proliferative proliferative (E (E22)) and and secretory secretory (E(E22P P44)) phases phases ofof the the control controlgroup; group;lower lowerpanel panel shows shows LGR5 LGR5 expression expression in both in both phases phases of of the the endometriosis endometriosis group group (E2 :(E 2: estradiol; estradiol; P4 : Pprogesterone). 4: progesterone). * p < 0.05. 2.1.3. Flow Cytometry Analysis 2.1.3. Flow Cytometry Analysis In order to determine the variation throughout the cycle, we next performed flow cytometry In order to determine the variation throughout the cycle, we next performed flow cytometry analysis to determine the percentage of LGR5+ cells+ in eutopic and ectopic endometrium. We measured analysis to determine the percentage of LGR5 cells in eutopic and ectopic endometrium. We a mean of 5.9% LGR5+ cells (range: 2–7%) from each sample. In the preliminary study, no variation in measured a mean of+ 5.9% LGR5+ cells (range: 2–7%) from each sample. In the preliminary study, no percentage of LGR5 cells was observed throughout the menstrual cycle in nine eutopic endometriosis variation in percentage of LGR5+ cells was +observed throughout the menstrual cycle in nine eutopic samples. Interestingly, no variation in LGR5 cells across the menstrual cycle was observed (Figure 3A). endometriosis samples. Interestingly, no variation in LGR5+ cells + across the menstrual cycle was Moreover, no significant differences were found between LGR5 cells in eutopic endometrium of observed (Figure 3A). Moreover, no significant differences were found between LGR5+ cells in women with endometriosis (differentiated by different types of disease) (Figure 3B) and control women eutopic endometrium of women with endometriosis (differentiated by different types of disease) (Figure 3C). However, comparison between ectopic lesions and eutopic endometrium in patients with (Figure 3B) and control women (Figure 3C). However, comparison between ectopic lesions and endometriosis (ovarian and DIE) revealed a significant increase of LGR5+ cells in ectopic lesions eutopic endometrium in patients with endometriosis (ovarian and DIE) revealed a significant (Figure 3D). These comparisons were only performed for ovarian endometriosis and DIE due to the increase of LGR5+ cells in ectopic lesions (Figure 3D). These comparisons were only performed for ovarian endometriosis and DIE due to the lack of ectopic samples from pelvic endometriosis (n = 1) Int. J. Mol. Sci. 2019, 20, 22 6 of 12 Int. J. Mol. Sci. 2018, 19, x 6 of 13 lack of ectopic samples from pelvic endometriosis (n = 1) and adenomyosis (n = 2). A t-test showed that and DIE adenomyosis lesions (n a= significantly contained 2). A t-test showed higher that DIE lesions percentage contained of LGR5 a significantly + cells than higher(Figure ovarian lesions percentage 3E). of LGR5+ cells than ovarian lesions (Figure 3E). Figure Figure 3. 3. Percentage Percentage of of FACS-sorted FACS-sorted LGR5 LGR5 + cells. (A) Percentages of LGR5 + cells. (A) Percentages of LGR5+ cells + cells in different phases in different phases ofofthe thecycle cycle(proliferative: (proliferative:nn == 9; 9; secretory: secretory: nn==4;4;ovulatory: ovulatory: n =n12; ND:ND: = 12; n=n9).=(B)9).Percentage of LGR5 (B) Percentage of + cells in LGR5 + eutopic cells endometrium in eutopic of different endometrium typestypes of different of endometriosis and controls of endometriosis (ovarian: and controls n = 8; npelvic: (ovarian: = 8; n = 3;nadenomyosis: pelvic: = 3; adenomyosis:n = 4; n =DIE: n = n10; 4; DIE: control: = 10; n =n12). control: (C)(C) = 12). Percentage PercentageofofLGR5 LGR5+ +cells cellsidentified identifiedin ineutopic eutopicendometrium endometriumofofwomenwomenwith withand andwithout withoutendometriosis endometriosis(control: (control:n n= =12;12; endometriosis: endometriosis: n= n=25). 25).(D) (D)Differences DifferencesininLGR5 LGR5++cells cells between between eutopic and ectopic eutopic and ectopicendometrium. endometrium.Left Leftpanel: panel:ovarian ovarian endometriosis(eutopic: endometriosis (eutopic:nn==8;8;ectopic: ectopic: nn == 4; 4; pp = = 0.0286). 0.0286). Right Rightpanel: panel:DIE DIE(eutopic: (eutopic:n n= =10;10; ectopic: ectopic:n= n=6; 6; p =p 0.0411). = 0.0411).(E)(E) Difference Difference between between ovarian ovarian and and DIE DIEectopic ectopicendometrium endometrium (ovarian: (ovarian: n =n 4; DIE: = 4; n= DIE: n=6; 6; p =p 0.0381). = 0.0381).ND: ND: non-determined; non-determined; DIE: deep DIE: deep infiltrating infiltratingendometriosis. endometriosis. * p*