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. 2014 Dec 29;100(3):E433–E442. doi: 10.1210/jc.2014-3720

Decreased Notch Pathway Signaling in the Endometrium of Women With Endometriosis Impairs Decidualization

Ren-Wei Su 1, Michael R Strug 1, Niraj R Joshi 1, Jae-Wook Jeong 1, Lucio Miele 1, Bruce A Lessey 1, Steve L Young 1, Asgerally T Fazleabas 1,✉
PMCID: PMC4333047  PMID: 25546156

Abstract

Context:

Endometriosis is a common gynecological disease affecting one in 10 women of reproductive age and is a major cause of pelvic pain and impaired fertility. Endometrial stromal cells of women with endometriosis exhibit a reduced response to in vitro decidualization. NOTCH1 is critical for decidualization of both mouse and human uterine stromal cells.

Objective:

This study aimed to determine whether decidualization failure in women with endometriosis is a consequence of impaired Notch signaling.

Setting and Design:

We investigated expression levels of Notch signaling components in the endometrium of women and baboons with or without endometriosis. We identified NOTCH1-regulated genes during decidualization of human uterine fibroblast (HuF) cells by microarray and quantified their expression levels in in vitro–decidualized endometrial stromal cells isolated from women with or without endometriosis.

Results:

Notch signaling receptors NOTCH1 and NOTCH4, ligands JAGGED2 and DLL4, as well as direct target genes HES5 and HEY1 were decreased in the eutopic endometrium of women and baboons with endometriosis. Notch signaling was decreased in stromal cells isolated from women with endometriosis, which was associated with impaired in vitro decidualization. Genes that were down-regulated by NOTCH1 silencing in decidualized HuF cells were also decreased in decidualized endometrial stromal cells of women with endometriosis. FOXO1 acts as a downstream target of Notch signaling and endometriosis is associated with decreased expression of NOTCH1-regulated, FOXO1-responsive genes during decidualization.

Conclusions:

Decreased Notch signaling is associated with endometriosis and contributes to impaired decidualization through the down-regulation of FOXO1.

Endometriosis is a common gynecological disease affecting 10–15% of women of reproductive age (1) and is characterized by the development of hormonally responsive endometrial glands and stroma outside the uterine cavity (2). Endometriosis is a major cause of pelvic pain and profoundly affects fertility: up to 50% of women with endometriosis are infertile compared with 5–10% of women without disease (3). In addition, women with endometriosis display a reduced monthly fecundity rate (2–10% compared with 15–20%) (3). Previous studies have shown that aberrant gene expression in the eutopic endometrium of women with endometriosis may contribute to disease-based implantation failure and infertility (4, 5). Endometrial stromal cells from women with endometriosis exhibit a reduced response to in vitro decidualization, which further supports the theory that abnormalities within the endometrium are responsible for subfertility caused by the disease (6, 7).

In the presence of estrogen and progesterone, human endometrial stromal cells surrounding the spiral arteries undergo “predecidualization” during the mid-to-late secretory phase of the menstrual cycle, which will persist and spread to the entire endometrial stroma if pregnancy ensues (8). During decidualization, stromal fibroblasts transdifferentiate into large, epithelioid-like decidual cells. Decidualized stromal cells secrete various proteins, including prolactin (PRL) and insulin growth factor binding protein 1 (IGFBP1) (9), which are considered markers of decidualization. Failure to mount an appropriate decidualization response is thought to be a cause of infertility, subfertility, or recurrent miscarriages (10).

Notch signaling is a highly conserved pathway across species, present in most multicellular organisms. It plays important roles for cell-cell communication, involving gene regulatory mechanisms controlling multiple cellular differentiation processes during embryonic and adult life (11). Notch signaling is mediated by four transmembrane receptors (Notch 1–4), and five transmembrane ligands of the Jagged/Delta-like families (11). Activation of Notch signaling is generally initiated through a juxtacrine mechanism, where adjacent cells expressing receptor and ligand interact, resulting in a series of receptor cleavage events, and the release of the Notch intracellular domain (NICD). Subsequently, the cleaved NICD translocates to the nucleus, where it binds and activates the Notch family transcription factor recombination signal binding protein Jκ, thereby initiating expression of Notch target genes, such as the Hairy enhancer of split (Hes) and Hes-related (Hey) transcription factor families (11). Nuclear and cytoplasmic noncanonical Notch signaling pathways have been described in a number of models. These include among others NICD-mediated activation of AKT via mTOR Complex 2 (mTORC2), possibly on the mitochondrial surface and activation of nuclear factor κB and estrogen receptor α via IκB-kinase family kinases in the cytoplasm and nucleus (12).

Our laboratory has previously shown that Notch1 modulates multiple signaling mechanisms critical for decidualization, using both a Pgr-cre conditional null mouse model as well as NOTCH1-silenced human uterine fibroblast (HuF) cells (13, 14). Given the possible role of Notch signaling in initiating the decidual response, we hypothesized that altered Notch signaling contributes to impaired decidualization in patients with endometriosis. In the present study, we demonstrate that the Notch signaling pathway is broadly down-regulated in the eutopic endometrium of women with endometriosis, as well as in baboons with spontaneous endometriosis. We have identified a group of NOTCH1-regulated genes during in vitro decidualization, which were decreased in decidualized endometrial stromal cells from women with endometriosis. Altogether, we provide evidence that Notch signaling is decreased in women with endometriosis, resulting in impaired decidualization.

Materials and Methods

Patient sample collection

Collection of eutopic endometrium using timed endometrial biopsies was carried out with the approval of Institutional Review Boards of the University of North Carolina (Chapel Hill, NC), the Greenville Health System (Greenville, SC), Michigan State University (East Lansing, MI), and Spectrum Health System (Grand Rapids, MI). Eutopic endometrium was obtained from women with laparoscopically confirmed endometriosis (EME) and women without disease (EM) ranging from 18–49 years of age. None of the women enrolled onto the study were on any type of hormonal therapy for at least 3 months prior to tissue collection.

Primary eutopic endometrial stromal cells were isolated from endometrial biopsies collected from control women (sc-EM) and women with endometriosis (sc-EME) at both the University of North Carolina (Chapel Hill, NC) and the Greenville Hospital System (Greenville, SC). Biopsies were cut to small pieces and then subjected to the same protocol as described for the isolation and culture of HuF cells (15).

Stromal fibroblasts from the decidua parietalis maintain a proliferating population of nondifferentiated fibroblastic cells, which closely resemble endometrial stromal cells (15). Human uterine fibroblast (HuF) cells were isolated from the decidua parietalis dissected from the placental membrane after normal-term vaginal delivery. Isolation and culture of HuF cells were completed as previously described (15). Cells isolated from each patient were grown individually and maintained for study between passages 3–5. Placental tissues were obtained with informed consent using a protocol approved by the institutional review board at Michigan State University and Spectrum Health System.

Demographic information for all patients included in this study is summarized in Supplemental Table 1.

Baboon sample collection

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois, Chicago, and Michigan State University. Endometrium from baboons with or without spontaneous endometriosis was collected by laparotomy during the midsecretory phase and spontaneous endometriosis was confirmed by laparoscopic presence of endometriotic lesions.

In vitro decidualization

Cells were treated with a hormonal cocktail of EPC (estrogen, progesterone, and cAMP) or vehicle for 6–8 days to induce in vitro decidualization. More details are provided in Supplemental Materials and Methods.

Lentivirus packaging and transfection

Lentivirus-based short-hairpin RNA (shRNA) plasmids targeting hNOTCH1 were purchased from Open Biosystems (Thermo) were packaged using Trans-Lentivial Packaging Kit (TLP5912, Thermo) in 293FT cell line (Invitrogen) according to the manufacturer's instructions. See Supplemental Materials and Methods for details.

Microarray and analysis

Four experimental analyses were performed: Group 1, transfected with empty plasmid; Group 2, transfected with hNOTCH1 lentiviral-shRNA plasmid; Group 3, transfected with empty plasmid and treated with EPC cocktail; and Group 4, transfected with hNOTCH1 lentiviral-shRNA plasmid and treated with EPC cocktail. RNAs were isolated, amplified to cRNA, and hybridized to Human U133-Plus 2.0 Array (Affymetrix). The array data was submitted to the Gene Expression Omnibus (GEO) database with the accession number GSE61827. See Supplemental Materials and Methods for details.

RNA isolation and real-time qPCR

See Supplemental Materials and Methods.

Immunohistochemistry

See Supplemental Materials and Methods.

Statistical analysis

Differences in mRNA expression between control and women with endometriosis were compared following normalization against RPL17. For data containing more than two groups, a nonparametric Kruskal-Wallis one-way ANOVA was used to test the null hypothesis of group differences, followed by a Wilcoxon test. For data with only two groups, a nonparametric Mann-Whitney U test was used. P < .05 were considered statistically significant. All statistical analysis was performed by GraphPad Prism 5.0 (GraphPad Software).

Results

Eutopic endometrium from women with endometriosis shows attenuation of the Notch signaling pathway

To detect mRNA levels of Notch signaling components, we performed qPCR on endometrial RNA isolates from women with or without endometriosis (midsecretory phase, EM and EME, respectively). We found that the expression of NOTCH1 and NOTCH4 were significantly lower in EME than EM (Figure 1A), whereas the other two Notch receptors, NOTCH2 and NOTCH3 were unchanged (data not shown). Of the five ligands that activate Notch signaling, JAG2 and DLL4 were significantly decreased in EME compared with EM (Figure 1B). Further, the Notch pathway target genes, HES5 and HEY1, were down-regulated in EME (Figure 1C).

Figure 1.

Figure 1.

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mRNA expression of Notch pathway signaling components in the eutopic endometrium of women or baboons with endometriosis. qPCR analysis of mRNA levels of two receptors (A, NOTCH1 and NOTCH4), two ligands (B, JAG2 and DLL4), and two target genes (C, HES5 and HEY1) that were decreased in the endometrium of women with endometriosis (EME; n = 10) compared with women without disease (EM; n = 8); In baboons, mRNA levels of receptors (D), ligands (E), and target genes (F) showed lower expression levels in endometrium of baboons with spontaneous endometriosis (Spont Eosis) compared with controls without disease (Con). *, P < .05; **, P < .01.

Immunolocalization using immunohistochemistry for NOTCH1, NOTCH4, and HES5 were performed on sections of EME and EM tissues (n = 6 in each group). NOTCH1 was highly expressed in both epithelial cells and stromal cells of EM, whereas in EME, NOTCH1 expression was dramatically decreased in both cell types (Figure 2A, first row). NOTCH4, both a Notch pathway receptor and transcriptional target gene of NOTCH1 (11), displayed a similar staining pattern as NOTCH1. Strong NOTCH4 staining was found on both epithelial and stromal cells of EM and significantly decreased in EME sections (Figure 2A, second row). The decrease in Notch staining was correlated with a decrease in the target gene HES5 (Figure 2A, third row). A Digital H-score method was used to analyze the expression levels of these proteins (Figure 2B) as previously described (16). Nonspecific rabbit IgG was used as a negative control and showed no immunostaning (Figure 2A, fourth row).

Figure 2.

Figure 2.

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Immunolocalization of Notch pathway signaling components in the eutopic endometrium of patients with endometriosis. Localization of NOTCH1, NOTCH4, and HES5 proteins were detected by immunohistochemistry in the endometrium of women with endometriosis (EME; n = 6) compared with women without disease (EM; n = 6). 3,3′ diaminobenzidine (DAB; brown) shows positive staining, normal rabbit IgG is used as negative control. Staining intensity of each section was quantified by image analysis software imageJ (D-HScore) and is shown in panel B. **, P < .01; bar = 50 μm.

Notch signaling is decreased in eutopic endometrium of baboons with spontaneous endometriosis

Only menstruating primates, such as the baboon, develop spontaneous endometriosis and the ectopic lesions closely resemble those that are found in women (2). We analyzed the expression of Notch signaling in the baboon endometrium from animals with or without spontaneous disease. In the eutopic endometrium of baboons with spontaneous endometriosis, the receptors NOTCH1 and NOTCH4 (Figure 1D), ligands JAG2 and DLL4 (Figure 1E), and target genes HES5 and HEY1 (Figure 1F) of Notch signaling all had significantly lower expression levels compared with control baboons without disease. These results are consistent with the pattern of expression seen in women with the disease.

Decreased Notch signaling in stromal cells from women with endometriosis is associated with impaired in vitro decidualization

Endometrial stromal cells from women with (sc-EME) or without (sc-EM) endometriosis were cultured and induced to decidualize in vitro. qPCR results confirmed that NOTCH1 and NOTCH4 receptor mRNAs that were down-regulated in whole endometrium of EME were also decreased in sc-EME compared with sc-EM (Figure 3A). In addition, JAG1, JAG2, and DLL4 were significantly decreased together with HES5 in sc-EME (Figure 3, B and C). This decrease in the Notch signaling pathway was correlated with a reduced decidual response, as determined by significantly decreased decidual marker gene expression of IGFBP1 and PRL (Figure 3, D and E).

Figure 3.

Figure 3.

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Notch pathway signaling component mRNA levels in decidualized endometrial stromal cells from patients with or without endometriosis is associated with a decrease in the decidualization markers IGFBP1 and Prolactin. mRNA levels of receptors (A, NOTCH1 and NOTCH4), ligands (B, JAG1, JAG2 and DLL4), and target genes (C, HES5 and HEY1) of Notch pathway signaling were decreased in decidualized stromal cells from endometrium of women with endometriosis (sc-EME; n = 6) compared with women without disease (sc-EM; n = 6). Marker genes of human decidualization IGFBP1 (D) and PRL (prolactin, E) also showed significantly lower expression in sc-EME. EPC/CON: expression level from decidualized stromal cells was normalized to expression of undecidualized cells from the same patient. *, P < .05.

Identification of NOTCH1 target genes during in vitro decidualization

Microarray analysis was performed to identify genes that are regulated by NOTCH1 during in vitro decidualization. A shRNA plasmid that targets human NOTCH1 or a control vector was first transfected into HuF cells via a lentivirus method. Next, cells were treated with the EPC cocktail or vehicle for 6 days to induce in vitro decidualization. Data were then analyzed and differentially expressed genes were selected based on a fold change greater than 1.5 in both sets of HuF cells, from the four different comparisons shown graphically in Figure 4A. Comparison 1 compares differential gene expression of decidualized vs nondecidualized HuF cells. We found 2447 genes significantly changed during in vitro decidualization in this comparison with 1036 genes down-regulated and 1411 genes up-regulated. Comparison 2 shows genes that were affected by NOTCH1 silencing in HuF cells that were induced to decidualize. Following NOTCH1 silencing, 45 genes were significantly down-regulated and 141 genes were significantly increased. In comparison 3, we compared the gene expression profile of NOTCH1 silenced HuF cells without EPC treatment to that of control cells without EPC treatment to identify NOTCH1-regulated genes in the absence of decidualization. Fifty-four genes were up-regulated genes and 59 were down-regulated in comparison 3. Comparison 4 identifies differentially expressed genes between decidualized and nondecidualized HuF cells under condition of NOTCH1 silencing. There are 791 down-regulated genes and 1192 up-regulated genes in this comparison. Overlap of differentially expressed genes in all four comparisons were calculated and represented in a Venn diagram (Figure 4B). We have identified 117 NOTCH1-regulated genes during decidualization by compiling significantly altered genes from both comparisons 1 and 2, whereas excluding genes changed as a result of silencing of NOTCH1 in the absence of decidualization (comparison 3), (Figure 4B, yellow zones, 26 and 91 genes, listed in Supplemental Table 2). Of these 117 genes, 39 genes were highly expressed as a consequence of in vitro decidualization but were down-regulated by NOTCH1 silencing, whereas another group of 68 genes which had low expression response to decidualization were up-regulated following NOTCH1 silencing. These two groups of genes comprise 90.6% of the whole gene list that NOTCH1 targets during in vitro decidualization, including IGFBP1, a marker of decidualization. However, another well-known decidualization marker PRL is increased in comparison 1 but not significantly changed in comparison 3 in the microarray. However, by qPCR analysis PRL expression was decreased following in vitro decidualization of NOTCH1 silenced HuF cells compared with controls (Supplemental Figure 1B). These results are consistent with our previous findings that silencing of NOTCH1 decreases the response of HuF cells to in vitro decidualization (Figure 4C). The array data was submitted to GEO database, accession number GSE61827.

Figure 4.

Figure 4.

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Determination of NOTCH1-regulated genes during in vitro decidualization of HuF cells. A, The four physiologically relevant comparisons used to identify genes regulated by NOTCH1 silencing and/or in vitro decidualization: comparison 1, vehicle vs EPC-treated (decidualized) HuF cells; comparison 2, decidualized (EPC) HuF cells without vs with NOTCH1 silencing; comparison 3, nondecidualized (Veh) HuF cells without vs with NOTCH1 silencing. Differentially expressed genes were selected using two-sample comparison by >1.5-fold change. B, Venn diagrams demonstrating the relationship between genes modulated in undecidualized HuF cells in response to NOTCH1 silencing. The green circle indicates genes selected by comparison 1, the red circle shows genes selected by comparison 2, the blue circle by comparison 3, and the gray circle by comparison 4. The numbers within the intersections of the circles indicate the common genes by two, three, or all four comparisons. The number below the comparison number indicates the total number of genes selected by that comparison. C, Cluster analysis of NOTCH1-regulated genes during decidualization, 117 genes selected by both comparisons 1 and 2 but not 3 were clustered in dimension according to their gene expression pattern by hierarchical-tree algorithm. The color code for the signal strength in the classification scheme is shown in the box at the bottom, in which up-regulated genes are indicated by red and down-regulated genes are indicated by blue.

NOTCH1-regulated genes are decreased in endometrial stromal cells from women with endometriosis

After identifying decidualization-specific genes' effect following NOTCH1 gene silencing, we sought to answer the question of whether stromal cells from patients with endometriosis displayed similar gene dysregulation. To answer this question, we selected and investigated 19 of 39 genes highly expressed during in vitro decidualization, which were down-regulated by NOTCH1 silencing from microarray data, based on previous reports suggesting that these genes relate to NOTCH1, decidualization, or endometriosis. Regulation of these genes by NOTCH1 silencing during in vitro decidualization was verified by qPCR (Supplemental Figure 2). Expression levels of the selected genes in endometrial stromal cells of patients with or without endometriosis were measured by qPCR. As a result, we were able to validate the decrease in 10 of these 19 genes in decidualized stromal cells from patients with endometriosis (ANGPTL4R, RORB, SOCS2, BCL2L11, BOC, CXCL12, C3, CFD, KCND2, and IGFBP1; Table 1). Of the other nine genes, six of them (FNBP1, FOXP1, GREB1, TNFSF10, MYOCD, and LEFTY2) showed a greater than 1.5-fold mean decrease; however, they failed to achieve statistical significance as assessed by Mann-Whitney U test. The decreased expression of NOTCH1-regulated genes in stromal cells from patients with endometriosis suggests that both decreased NOTCH1 and Notch pathway signaling significantly contributes to the inhibition of decidualization as a consequence of the disease.

Table 1.

qPCR Analysis of NOTCH1 Target Genes in Decidualized Endometrial Stromal Cells of Women with or without Endometriosis

Gene Fold Change
 P Value
EPC/CTRL in sc-EM EPC/CTRL in sc-EME sc-EME/sc-EM
ANGPTL4a 3.317 −1.835 −6.098 .008
BCL2L11a 6.013 2.683 −2.242 .016
BOCa 2.542 0.948 −2.681 .008
C3a 20.131 4.909 −4.098 .008
CFD 116.224 13.099 −8.850 .056
CXCL12a 3.701 0.558 −6.623 .008
FNBP1 2.209 1.042 −2.119 .278
FOXP1 2.110 1.171 −1.802 .095
GREB1 4825.390 2572.191 −1.876 .278
IGFBP1a 420 330.518 91 865.317 −4.566 .029
IL-15 11.641 29.190 2.508 .452
KCND2a 19.117 2.596 −7.353 .008
LEFTY2 174 513.070 3136.879 −55.556 .171
MYO10 2.319 1.808 −1.282 .206
MYOCD 17.873 8.524 −2.096 .143
PDZD2 1.761 3.959 2.248 .452
RORBa 254.757 78.099 −3.257 .032
SOCS2a 2.880 1.324 −2.174 .032
TNFSF10 14.975 7.545 −1.984 .095
PRLa,b 3354.306 529.570 −6.329 .029
FOXO1a,b 43.426 16.185 −2.681 .029
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Primary eutopic endometrial stromal cells were isolated from endometrial biopsies collected from sc-EM and sc-EME, and then treated by EPC to induce in vitro decidualization. Gene expressions in EPC cells from each individual patient were normalized by CTRL cells from the same patient (EPC/CTRL). Gene expression in sc-EM and sc-EME are listed in columns 2 and 3, fold changes of these genes in sc-EME compare with sc-EM are listed in columns 4, minus means gene is decreased in sc-EME compared with sc-EM.

a

P < .05.

b

These two genes are not selected from microarray, but show significant decrease in NOTCH1-silenced HuF cells by qPCR (Supplemental Figures 1 and 2, respectively).

FOXO1 is a downstream target of Notch signaling

IGFBP1 (17), BCL2L11, and LEFTY2 (18), are target genes of forkhead transcriptional factor FOXO1, and we have identified that these genes are down-regulated by NOTCH1 silencing along with their decreased expression in endometrial stromal cells of women with endometriosis. Because Foxo1 is decreased in the decidua of Notch1 knockout mice (13), we hypothesized that FOXO1 could be a downstream target of NOTCH1. To prove our hypothesis, we first confirmed regulation of FOXO1 by NOTCH1 silencing in HuF cells. By qPCR and Western blot, we found that FOXO1 was decreased after NOTCH1 was silenced by shRNA in in vitro decidualized HuF cells (Figure 5, A and B; Supplemental Figure 2). Next, we detected expression of FOXO1 in endometrial stromal cells of endometriosis patients and found that FOXO1 expression levels were lower in patients with endometriosis than in women without disease (Table 1).

Figure 5.

Figure 5.

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FOXO1 acts downstream of NOTCH1 during endometriosis-related decidualization impairment. Both mRNA (A) and protein (B) levels of FOXO1 in in vitro decidualized HuF cells were decreased by NOTCH1 silencing. In decidualized endometrial stromal cells from women with endometriosis (sc-EME), FOXO1 mRNA showed lower expression compared with women without disease (sc-EM). (EPC/CON: expression level from decidualized stromal cells was divided by expression of undecidualized cells from same patient to normalize; *, P < .05). C, Working model: Notch signaling (mainly NOTCH1) is decreased in eutopic endometrium of women with endometriosis, and cleavage of NOTCH1 protein is altered because of disease-related progesterone resistance. Attenuation of NOTCH1 contributes to impaired decidualization of endometrial stromal cells from women with endometriosis via its downstream target FOXO1 and FOXO1 target genes. #, (14); *, (37, 40).

Discussion

The uterus is a dynamic physiological system in which cellular proliferation, terminal differentiation, and apoptosis occur in a cell- and time-specific manner under regulation of ovarian and embryonic hormones during the menstrual cycle and pregnancy (19, 20). According to one of the most widely accepted theories, endometriosis develops from endometrial fragments disseminated into the peritoneal cavity via retrograde menstruation, which are able to survive, implant, and invade to establish endometriotic lesions (21, 22). The percentage of infertile patients in the population of women with endometriosis is much higher than women without disease (3). Decidualization is a process in which estrogen-primed endometrial stromal cells differentiate into secretory, epithelioid decidual cells in response to postovulatory increase of ovarian progesterone (9). The decidua critically regulates trophoblastic invasion and placentation (23). Impaired decidualization leads to infertility, subfertility, or recurrent miscarriages (10). To date, there are several studies indicating that endometrial stromal cells from women with endometriosis display an impaired decidualization response (4–7, 24), but the mechanism underlying this limited response to in vitro decidualization is still unclear. Therefore, understanding mechanisms underlying impaired decidualization in women with endometriosis will be critical for advancing future work in the clinical management of fertility in endometriosis patients.

Notch signaling mediates cell fate decisions, proliferation, and cell-cell communication via interactions with neighboring cells in a juxtacrine manner (25). Previously, we showed that absence of Notch1 in the mouse uterus leads to impaired decidualization via multiple mechanisms (13) and shRNA-mediated NOTCH1 silencing causes lower levels of IGFBP1 and IL-11, two markers of human decidualization (14). In contrast, overexpression of JAG1, a Notch ligand, in endometrial stromal cells induces a wide spectrum of genes including IGFBP1 (26). Of the 117 NOTCH1-regulated genes reported in our study, 12 were the same as those reported to be regulated as a consequence of JAG1 overexpression. Moreover, we show that multiple Notch signaling components, including NOTCH1, were significantly decreased in women with endometriosis as well as in baboons with spontaneous disease. Impaired response to in vitro decidualization of stromal cells isolated from the endometrium of endometriosis patients in our study was consistent with previous studies (6, 7). Taken together, these data lead us to conclude that decreased Notch signaling contributes to impairment of decidualization in the eutopic endometrium of women with endometriosis. Further, silencing of NOTCH1 by shRNA affects decidualization at the initiation of hormone treatment (Supplemetal Figure 1). A majority of target genes of NOTCH1 during in vitro decidualization identified by microarray were also decreased in decidualized stromal cells from women with endometriosis. Altogether, our data clearly demonstrates that NOTCH1 loss of function plays a key role in endometriosis-related decidualization impairment of stromal cells.

FOXO1 is one of the earliest genes induced during decidualization (27). The importance of FOXO1 for the induction of decidualization has been conclusively demonstrated in a number of in vitro experiments (28–30). Overexpression of FOXO1 in human endometrial stromal cells can transcriptionally increase expression of decidualization markers IGFBP1 and PRL independent of hormones and cAMP (17, 29, 31, 32). In addition, FOXO1 can act as a coactivator of Notch signaling by directly interacting with recombination signal binding protein Jκ, to recruit coactivators to the promoters of Notch target genes, which in turn regulates the differentiation of several cell types (33). This functional NOTCH1-FOXO1 axis is necessary for myogenesis (33), a process of transdifferentiation analogous to decidualization. We show here that in human endometrium, NOTCH1 regulates the levels of FOXO1 mRNA, directly or indirectly. Therefore, we propose that NOTCH1 regulates FOXO1 expression, which in turn induces decidualization, perhaps through a NOTCH1-FOXO1 axis similar to the one required for myogenesis. Because FOXO1 is also decreased in the endometrium of women with endometriosis (34–36), attenuation of NOTCH1 signaling in endometriosis therefore inhibits the critical initiation pathways required for the process of decidualization. Furthermore, in our microarray analysis, we identified some FOXO1-dependent genes, important for decidualization, that were suppressed in response to NOTCH1 silencing. These included IGFBP1, P2RY14, BCL2L11, LEFTY2, SLC40A1, KIA0101, RRM2, ANLN, PRC1, PSAT1, ZWINT, SLC7A1, and ZWILCH (29). This further supports our data, which suggests that decreased NOTCH1 resulting from endometriosis prevents the induction of FOXO1 in endometrial stromal cells, which in turn inhibits the decidualization response.

Progesterone resistance is associated with the dysregulation of progesterone-dependent gene networks in the endometrium of patients with endometriosis (37), which was further confirmed in our baboon endometriosis model (38–40). Our previous data showed that progesterone activates Notch pathway signaling via increased cleavage of NOTCH1 (14), suggesting that progesterone resistance in the endometrium of patients with endometriosis partly contributes to the decreased Notch signaling. FOXO1 can interact with the progesterone receptor to coordinate cell cycle regulation and differentiation of human endometrial stromal cells. Collectively, our data suggest that during normal decidualization, progesterone receptor together with NOTCH1 regulates FOXO1 expression. However, as a consequence of endometriosis, progesterone resistance causes decreased Notch pathway activity, resulting in decreased FOXO1 and a subsequent impaired decidualization response.

In summary, our study identifies an important role of Notch signaling during the initial phase of decidualization, a process that is vital for successful implantation and the establishment of pregnancy in women. Our data, for the first time, clearly demonstrates that Notch signaling was significantly decreased in eutopic endometrium of women and baboons with endometriosis compared with disease-free controls. This decrease in Notch signaling was further confirmed in in vitro–decidualized endometrial stromal cells from women with endometriosis, which also showed significantly compromised decidualized response compared with decidualized stromal cells from disease-free women. Our data strongly support a model in which altered Notch signaling contributes to impaired decidualization. This effect is mediated at least in part by the decreased expression of the Notch downstream target and coactivator FOXO1 in women with endometriosis (Figure 5C). Identifying these crucial signaling pathways may help in designing better therapeutic approaches as well as helping improve the fertility in that subset of infertile women with endometriosis.

Acknowledgments

We thank the Human Female Reproductive Tract Biorepository and the Spectrum Health Medical Group, Department of Obstetrics, Gynecology and Reproductive Biology (especially Elizabeth Leary, MD; Calvin Leazenby, MD; Christine Heisler, MD; Maryanne George, NMW; and the CORE Faculty: Melinda Johnson, MD; Michelle Backus Walzer, MD; Rebecca Caldwell, MD; and Darla Olson, MD) for their help in obtaining human samples for research use. We also thank Samantha Bond and Meighan McAuliffe (Michigan State University) for technical assistance, Mark Olson (Michigan State University) for assistance with the baboon studies, and Susan Ferguson (Michigan State University) for isolation of HuF cells from term placentas.

This work was supported by National Institutes of Health Grants RO1 HD42280 (to A.T.F.) and RO1 HD 067721 (to B.A.L. and S.L.Y.).

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
EM
women without disease
EME
women with laparoscopically confirmed endometriosis
EPC
estrogen, progesterone and cyclic adenosine monophosphate
FOXO1
Forkhead box protein O1
GEO
Gene Expression Omnibus
HuF
human uterine fibroblast
IGFBP1
insulin growth factor binding protein 1
NICD
Notch intracellular domain
PRL
prolactin
sc-EM
stromal cells of endometrial biopsies collected from control women
sc-EME
stromal cells of endometrial biopsies collected from women with endometriosis
shRNA
short-hairpin RNA.

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