Cabergoline Stimulates Human Endometrial Stromal Cell Decidualization and Reverses Effects of Interleukin-1β In Vitro

Context Human embryonic implantation is regulated by neuroendocrine hormones, ovarian steroids, growth factors, and cytokines. Sympathetic innervation of the uterus also may play a role.

We tested the hypothesis that cabergoline (Cb), an agonist of type 2 dopamine receptors (DRD2), could influence endometrial decidualization in vitro.

Immunohistochemistry confirmed the presence of catecholaminergic neurons in human uterine tissue. DRD2 mRNA and protein expression in endometrial tissue and cells were validated by quantitative RT-PCR, cDNA microarrays, RNA sequencing, and Western blotting. Isolated human endometrial stromal cells (ESC) were subjected to dose-response and time-course experiments in the absence or presence of decidualizing hormones (10 nM estradiol, 100 nM progesterone, and 0.5 mM dibutyryl cAMP). In some cases, interleukin (IL)-1β (0.1 nM) was used as an inflammatory stimulus. Well-characterized in vitro biomarkers were quantified.

DRD2 were maximally expressed in vivo in the mid-secretory phase of the cycle and upregulated in ESC in response to decidualizing hormones, as were classical (eg, prolactin) and emerging (eg, VEGF and connexin 43) differentiation biomarkers. Cabergoline treatment more than doubled decidual biomarker expression, whereas risperidone, a dopamine receptor antagonist, inhibited ESC differentiation by >50%. Cabergoline induced characteristic decidual morphology changes and blocked detrimental effects of IL-1β on decidual cytology.

Our results support the hypothesis that dopaminergic neurons modulate decidualization in situ. We postulate that dopamine agonists, like Cb, could be developed as therapeutic agents to enhance implantation in couples with inflammation-associated infertility.

uterus, dopamine receptors, differentiation, steroids, cytokines During vertebrate evolution, a subset of placental mammals developed spontaneous endometrial decidualization and menstruation as a reproductive strategy. These include 4 bat species, spiny mice, the elephant shrew, nonhuman old-world primates, and humans (1). A common feature underlying this seemingly inefficient procreative process (2) involves phasic proliferation, differentiation, and sloughing (in the absence of pregnancy) of the uterine mucosa as a result of fluctuating neuroendocrine hormones and ovarian steroids; in particular, a large excursion in luteal progesterone (P4) concentrations is critical. In women, 17β-estradiol (E2) and P4 alone can transform the endometrium into a highly vascularized and immune-privileged microenvironment that promotes embryo implantation (3, 4). These 2 ovarian steroids orchestrate endometrial receptivity via elaboration of local eicosanoids (5), growth factors (6), and vitamins (7) from divergent cell populations within the uterus. The complex endometrial cellular microenvironment comprises predominantly epithelial, stromal, endothelial, and immune elements. Reports that neurons innervate the human uterine lining date back to the 1940s. Dallenbach and Vonderlin (8) suggested that “it is possible that the nerves influence blood flow, cellular metabolism and secretory function.” More recently, sensory Aδ and C fibers associated with pain and dysmenorrhea (9) and adrenergic and dopaminergic nerves linked with vascular modulation (10, 11) have been recognized. The anatomically proximate relationship between neurons and capillaries led us and others (12) to hypothesize a common trophic mechanism that we coined neuroangiogenesis (13). Among the different types of endometrial nerves recruited into the uterine mucosa are catecholaminergic neurons that express tyrosine hydroxylase and synthesize dopamine (14). Although it was initially reported that dopamine had no direct action in endometrial cells (15) and that dopamine receptors were not expressed in primate endometrium (16), these signaling molecules have since been validated and localized in human endometrial cells (17, 18). The studies described in the current report were undertaken to test the hypothesis that neurotransmitters derived from endometrial neurons contribute to uterine decidualization and hence play a regulatory role in early pregnancy. We chose to examine the impact of the dopamine receptor D2 (DRD2) agonist cabergoline (Cb) on human endometrial stromal cell (ESC) differentiation using an established panel of validated biomarkers (19). Our overarching goal is to identify paracrine mediators of decidualization, as these molecules or their congeners have potential as interventions to promote uterine receptivity in couples with impaired fecundity (20). In addition to the discovery that some eicosanoids, growth factors and vitamins (7) enhance endometrial differentiation, investigators also have established that cytotoxic cytokines, particularly interleukin (IL)-1β, secreted from resident tissue macrophages (21), can cause intrauterine inflammation and perturb decidualization (22-24). Free serum IL-1β levels correlate inversely with clinical in vitro fertilization (IVF) implantation rates (25) and multivariable logistical regression of endometrial fluid cytokines showed a negative correlation between IL-1β concentrations and clinical IVF pregnancy rates (26). We have shown that IL-1β can disrupt the secretion of prolactin, insulin-like growth factor–binding protein 1, and other differentiation markers of human ESC in response to a standard endocrine stimulus comprising E2 (10 nM), P4 (100 nM), and dibutyryl cyclic adenosine monophosphate (cAMP) (0.5 mM) (27). In vitro exposure to IL-1β prevents the expected mesenchymal-to-epithelial transformation (MET) associated with decidualization (19) and recapitulates patterns of gene expression observed in vivo in the setting of endometriosis and infertility (28). The findings that we report in this paper demonstrate that Cb ligation of the DRD2 receptor, and possibly other neurotransmitter receptors, robustly induces biomarkers of ESC decidualization. By contrast, the DRD2 inhibitor risperidone reduces these biomarkers. Moreover, addition of Cb to ESC in vitro reverses the inhibitory effects of IL-1β on ESC differentiation. We postulate that supplementation of Cb, or possibly other DRD2 agonists, might stimulate ESC differentiation in vivo and ultimately could be useful as therapeutic agents to enhance endometrial receptivity in couples with inflammation-associated infertility or repetitive pregnancy loss.

Source of Human Tissues Healthy, parous women with regular menstrual cycles, who had not received hormonal therapy for at least 3 months prior to surgery, were recruited; however, nonsteroidal anti-inflammatory pain medications were allowed. The 5 subjects ranged in age from 30 to 35 years and had normal fasting serum prolactin levels (<20 ng/mL). Endometrial tissue specimens were obtained after providing written informed consent under institutional review board (IRB)-approved study protocols at Wake Forest School of Medicine (#00019887) to evaluate pelvic pain and pressure symptoms. In the current study, we included subjects who underwent indicated laparoscopy and were found to have no visible evidence of endometriosis or pelvic pathology with the exception of subserosal leiomyomas or paratubal cysts. Endometrial Pipelle biopsies were collected in the mid-proliferative phase to avoid effects of endogenous P4 and used for histological assessments and to prepare primary cell cultures as described below. The biopsies were collected under sterile conditions and transported to the laboratory on ice in DMEM/Ham’s F-12 (cat#10-092cv, CellGro, Manassas, VA) containing 10% fetal bovine serum (FBS). Serum P4 was not measured on the day of biopsy for menstrual phase confirmation, but histological dating (29) confirmed the clinical determination of the proliferative cycle phase in all 5 subjects. Immunohistochemistry Paraffin-embedded endometrial biopsy tissues were cut into 5-µm thick sections and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) as described previously (30). Briefly, after mounting, the sections were deparaffinized in xylene and rehydrated in graded concentrations of ethanol. Antigen retrieval was performed by heating the slides in 10 mM sodium citrate buffer (pH 6.0) at 100 °C for 4 minutes. Slides were exposed for 10 minutes to 3% hydrogen peroxide in methanol to quench endogenous hydrogen peroxidase activity; each slide was then extensively rinsed in water. Nonspecific binding was blocked with Super Block (cat# AAA-500, ScyTek Laboratories, Logan, UT) 30 minutes at room temperature. Sections were incubated with primary antibodies to tyrosine hydroxylase (catalog #NBP2-42211, Novus, Centennial, CO) or DRD2 (catalog #MAB9266, Novus), each diluted 1:100 in Tris buffer, pH 7.4, overnight at 4 °C. Antibodies and conditions for estrogen receptor (ER)α IHC were published previously (cat# 13258, Cell Signaling, 1:200 dilution, Danvers, MA) (31). Staining was performed using EnVision Plus Systems (cat# K406311-2 and K346811-2, Agilent, Santa Clara, CA) reagents, according to the manufacturer’s protocol, with mild counterstaining using Mayer’s hematoxylin. The negative staining control was performed by substituting the primary antibody with non-immune serum IgG diluted 1:200 (Biogenex, San Ramon, CA). Photomicrographs were captured on an EVOS Cell Imaging device. Establishment of Human ESC Cultures Independent human ESC cultures were prepared from proliferative phase biopsies from each of the 5 subjects. The cells were subcultured at least twice to eliminate contamination by macrophages or other leukocytes, as described previously (30). Each culture was studied before the sixth passage to avoid cellular de-differentiation (32). ESC cultures prepared by this protocol were >93% pure and retain phenotypic endometrial stromal markers in vitro, including functional estrogen and progesterone receptors (33). Cells were plated directly onto 10-cm polystyrene dishes without exogenous extracellular matrix at an initial plating density of 25 000 cells per cm2 and grown to 60% to 80% confluence in phenol red-free Roswell Park Memorial Institute (RPMI) 1640 medium without L-glutamine and phenol red supplemented with 5% charcoal-stripped (hormone-depleted) fetal calf serum. ESC Treatment With Hormones, Cabergoline, Risperidone, IL-1β and Kinase Inhibitor and Effects on In Vitro Decidualization To induce decidualization, ESC were subjected to our established differentiation protocol by incubation with 10 nM E2, 100 nM P4, and 0.5 mM dibutyryl cAMP (“hormones,” “H”) for up to 7 days in hormone-depleted media. Cabergoline was added to the culture media within a concentration range of 1 to 100 μM, based on published studies (34-36), as well as our own preliminary dose-response results. Risperidone, an antagonist of DRD2, was used at a concentration of 100 μM (37). IL-1β was studied at a final concentration of 0.1 nM, which we showed was the IC50 (the molar cytokine concentration required to achieve 50% inhibition) for prolactin, vascular endothelial growth factor (VEGF), and connexin 43 (Cx43) expression in ESC (27). The MEK1/2 inhibitor PD98059 (“PD”) was used at a final concentration of 15 μM, which we optimized in prior ESC studies as an effective inhibitor of IL-1β effects during ESC decidualization (27). RNA Isolation and Quantitative Real-Time-Polymerase Chain Reaction To verify the expression of DRD2 in endometrial stroma we prepared 5 independent ESC cultures and subjected them to quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The ESC were incubated without or with hormones for 48 hours as described above. Total RNA was isolated from the cells using TRI-reagent (Life Technologies) following the manufacturer’s protocol and frozen at −80 °C until analyzed. The cDNA was synthesized from mRNA samples and subsequently used as template for mRNA expression assays. For real-time qRT-PCR, we used the SsoAdvanced Universal SYBR Green Supermix reagent (cat #1725271, Bio-Rad Laboratories, Hercules, CA) and followed the vendor guidelines with some modifications and validated DRD2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (QIAGEN QuantiTect Primer Assay, cat #s QT00012558 and QT00079247, respectively, Toronto, ON). A total reaction volume of 20 µL contained 10 µL SYBR Green, primer mix 2 µL, 1 µL 50 mM MgCl2, 2 µL H2O and 5 µL cDNA. The PCR was run for 40 cycles in a CFX real-time thermocycler (Bio-Rad) and data were analyzed after normalization to GAPDH mRNA levels, using the formula 2–ΔΔct, where ct is the cycle threshold. cDNA Illumina Microarray Analyses Total RNA was isolated from ESC according to the manufacturer’s protocol (Qiagen RNeasy Mini Kit: Qiagen; Valencia, CA). RNA integrity was determined initially by 260/280 ratios = 1.9 to 2.1 and finally by scanning with an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip. Isolated RNAs were reverse transcribed to dsDNA and kept at −80 °C until microarray analysis. The Emory Integrated Genomics Core analyzed samples for gene expression using Illumina Whole Genome BeadChips (Illumina; San Diego, CA). Double-stranded cDNA libraries were prepared and loaded onto Illumina Human HT12 expression chips. Raw probe intensities were corrected for background levels and normalized by the quantile normalization algorithm using GenomeStudio software from Illumina. To restrict our analysis to probes significantly expressed in ESC samples, we removed probes that had detection P values < 0.05 for fewer than 10% of samples, resulting in a final set of 27 616 probes that were eligible for analysis. Secondary Analysis of Single Cell mRNA Data We analyzed publicly accessible data from single cell RNA sequencing of 2148 isolated cells from 19 endometrial biopsies (38). DRD2 mRNA transcript levels were quantified with respect to the menstrual cycle day. In samples with high sequencing depth, unciliated epithelial and stromal fibroblasts appeared to be the major endometrial cell sources of DRD2 transcripts. Western Blot Analyses Western blots were performed on whole-cell extracts obtained after scraping ESC into Eppendorf tubes, centrifugation, discarding the supernatant, and vortexing the pelleted cells in 100 to 200 µL extraction buffer (cat# FNN0011, Life Technologies, Grand Island, NY). Total proteins (30 μg per lane, determined using the Thermo Scientific-Pierce BCA Protein Assay kit, cat# PI-23227, Thermo-Fisher Scientific) were run on NuPAGE Novex 4-12% Bis-Tris Protein Gels, transferred to PVDF membranes and blocked with 5% skim milk in PBS+T. Specific Cx43, ERα, progesterone receptor (PR), and mitogen-activated protein ERK kinase (MEK-ERK)1/2 cascade phosphoproteins were detected using validated antibodies at optimized concentrations, based on our prior report (39) DRD2 antibodies (cat# AB85367, Abcam, Cambridge, MA), were used at a final concentration of 1:300. After overnight labeling with the primary antisera, blots were then incubated with a secondary goat anti-rabbit or anti-mouse antibody (1:300 000; cat#s 31460 and 31430, Pierce Biotechnology Inc., Rockford, IL) linked to horseradish peroxidase and immunoreactive bands were visualized by the Enhanced Chemiluminescence (ECL) System (Amersham Pharmacia Biotech, Piscataway, NJ). The size of each band is indicated as its corresponding molecular mass (kDa) in the figures. Quantification of the Western blot data was performed by laser densitometry; values were normalized with respect to the corresponding β-actin band density and reported as fold-changes relative to the untreated control values, expressed as mean ± standard error of the mean (SEM). Quantification of Cell Shape Index Cell morphology was assessed daily by phase-contrast microscopy. Micrograph images were photographed with a green filter to enhance clarity and measured using a modified calculation of “roundness” that we have described previously (19, 40), where a cell shape index of 1.0 = a maximally circular, decidualized cell. Ten cells were randomly captured under each hormone-, Cb-, or cytokine-treatment condition, representative of 3 independent subject-derived cultures, and they were measured for shape. Results are presented as mean cell shape index ± SEM. Enzyme-Linked Immunosorbent Assay Sensitive and specific commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits that we previously validated for ESC supernatants (19, 41) were used to quantify prolactin (Alpha Diagnostic International, San Antonio, TX), and VEGF (R&D Biosystems, Minneapolis, MN). As we and others have demonstrated, these are established markers of ESC differentiation (3, 19, 42). Protein and mRNA Quantification and Statistical Analyses The ELISA and Western blot data were performed in triplicate and the results are representative of experiments from a minimum of 3 cell preparations from independent subjects. Expression levels of the cellular or secreted proteins were found to be normally distributed by Kolmogorov-Smirnov tests and the data are presented as mean ± SEM (27). The qRT-PCR data were normalized to GAPDH transcript levels as described above. Comparisons were determined using analysis of variance (ANOVA) with post hoc Tukey’s tests for multiple inter-group differences or by paired Student’s t tests for 2-group analyses. In experiments where hormones and dopamine receptor agonists or antagonists were administered in combination, 2-way ANOVA methods and Tukey’s post hoc analyses were used. Statistics were done using R 3.6.2 (R Core Team, 2019, R Foundation for Statistical Computing, Vienna, Austria: https://www.R-project.org/.) Statistical significance for all the analyses was accepted when 2-tailed tests yielded P < 0.05 between the treatment groups. Numbers of biological replicates (n) are indicated in the Figure legends.

IHC Localization of Tyrosine Hydroxylase-Positive Nerve Fibers in Human Uterus To validate the presence and localize the distribution of catecholaminergic nerves in endometrium we performed IHC on paraffin-embedded tissue. As reported previously (11), evidence of these neurons was revealed by prominent, tyrosine hydroxylase–immunopositive fibers coursing through the myometrium and deep endometrial basalis (Fig. 1A) and penetrating into the superficial stroma between epithelial glands within the functionalis layer (Fig. 1B). Similar findings were confirmed in biopsies from 3 subjects. Histology and IHC Localization of DRD2 and ERα in Endometrial Biopsies Serial sections of endometrial tissue stained with anti-DRD2 monoclonal IgG antibodies identified the cytoplasm of endometrial stromal cells as a rich source of DRD2 (Fig. 2A), with faint epithelial cytoplasmic signal also noted. These qualitative findings are consistent with the publication of Novella-Maestre et al (18). The presence of nuclear ERα antigens in epithelial and stromal elements (Fig. 2B) served as a positive IHC control and as a comparison to the distribution of DRD2. Hematoxylin and eosin (H&E) staining delineated the characteristic morphology of proliferative endometrium, showing tubular glands and dense stroma (Fig. 2C). Negative controls using isotype-specific nonimmune serum IgG revealed no immunoperoxidase staining (Fig. 2D). DRD2 mRNA Transcripts and Protein Are Expressed in ESC and Both Are Upregulated by Hormones To validate the IHC findings that endometrial stromal cells express DRD2 in situ, we isolated mRNA from 3 independent ESC preparations and subjected it to qRT-PCR. Melting curve analysis showed characteristic, single peaks for DRD2 and GAPDH transcripts (data not shown), with the latter used as an internal, constitutive comparator. Exposure of ESC to decidualizing hormones (E2, P4, and cAMP) for 2 days afforded a 2.7 ± 0.8-fold increase in DRD2 transcripts (Fig. 3A, n = 3, Student’s t test, P < 0.05). Bioinformatics approaches also were used to corroborate the expression of endometrial DRD2 mRNA. Based on publicly accessible data from single cell RNA sequencing (-seq) analyses (38), DRD2 mRNA transcript levels were evaluated. From 19 healthy women sampled between cycle days 4 and 27, 2148 cells were recovered and sequenced. DRD2 mRNA concentrations were much higher on d22 of the menstrual cycle than the other cycle days (Fig. 3B). In samples with high sequencing depth, unciliated epithelial and stromal fibroblasts appeared to be the major endometrial cell sources of DRD2 transcripts. DRD1, DRD3, and DRD4 mRNAs were barely detectable by this single cell sequencing method. Illumina cDNA microarrays of the 5 ESC preparations, which use optimized probes to amplify target genes and are often more sensitive than RNA-Seq, confirmed that DRD2 mRNA was 3.3-, 4.7-, and 19.5-fold more abundant than DRD1, DRD3, and DRD4 transcripts (ANOVA, P < 0.05). Consistent findings were noted at the protein level, with progressive increases in DRD2 determined by Western blotting after 2 (H+2, 2.2 ± 0.2-fold) and 5 (H+5, 3.5 ± 0.2-fold) days exposure to decidualizing hormones (H) compared with controls (ANOVA P < 0.01, P < 0.05 for all 3 interactions by Tukey’s post hoc tests) (Fig. 4). These results indicate that hormones upregulate DRD2 expression, providing an endocrine mechanism for enhanced response to dopamine signaling in decidualized stromal cells. The MEK1/2 inhibitor PD98059 (PD) had little independent effect, alone or with hormones, on DRD2 concentrations (Fig. 4). β-Actin expression also was not affected. Morphological Effects of Cabergoline on ESC Cytology To evaluate the effects of Cb on ESC differentiation we first assessed changes in ESC cell morphology, which prior reports have shown is a sensitive indicator of differentiation status (19, 40) and has been recognized for decades (43). Using cell shape index determinations, we observed that Cb induced dramatic morphological changes within 48 hours (Fig. 5) that typically require at least 7 days with the classic hormone-induced decidualization protocol (19). Calculation of cell shape indices revealed differential effects of Cb and hormones (H). The findings are described in Table 1. It should be noted that the combination of hormones + Cb yielded cell shape indices of 0.77 ± 0.08 within 48 hours, (Table 1), approaching the results reported in our prior report of 0.83 ± 0.04 after 14 days exposure to E2, P4, and cAMP (19). Table 1. | ESC treatment (× 48 h) | Cell shape index | (±) SEM | Significant differences | |---|---|---|---| | Control | 0.38 | 0.03 | | | Cabergoline (Cb) | 0.61 | 0.09 | *, | | Hormones (H) | 0.45 | 0.05 | *, ** | | Cb + Hormones | 0.77 | 0.08 | *, *** | ANOVA P < 0.05 with Tukey’s post hoc tests (* differs from control, ** differs from Cb, *** differs from Hormones). DRD2 Receptor Activation Augments and DRD2 Blockade Inhibits Biochemical Markers of ESC Decidualization To assess potential physiological functions of DRD2 signaling in ESC, time-course experiments were performed by analyzing well-characterized biomarkers of ESC differentiation. As shown in Fig. 6A and 6B, prolactin and VEGF secretion increased progressively, as previously reported (27), following incubation with hormones for up to 5 days (H+5). When the dopamine receptor agonist Cb was co-incubated with hormones (Cb+H), additive effects were noted, particularly with respect to prolactin production (Fig. 6A). Two-way ANOVA of the prolactin ELISA data showed effects of days of incubation (F = 7.20, P < 0.01) and Cb and hormone treatments (F = 14.57, P < 0.001). Due to significant interactions among time and treatment factors, we could not perform post hoc analysis of 2-way ANOVA data, but 1-way ANOVA with Tukey’s post hoc analyses indicated that the 3 treatment conditions all differed significantly by day 2 of incubation (P < 0.05). Similar findings were noted for VEGF secretion, with statistically significant effects of incubation time (F = 11.61, P < 0.001) and treatments (F = 15.27, P < 0.001). One-way ANOVA with Tukey’s post hoc analyses indicated that the 3 treatment conditions all differed significantly on days 2 and 3 of incubation (P < 0.05), whereas hormones and Cb+H differed from controls on day 5 (P < 0.001), but not from each other (P = 0.53). ESC incubated with Cb in the absence of hormones had undetectable prolactin (data not shown), but VEGF secretion from the ESC cultures was doubled 3 days after Cb treatment, even in the absence of hormones (Fig. 6C, Student’s t test, n = 3, P < 0.05). To evaluate other stimulatory interactions of Cb and hormones, we examined an aggregate of decidual biomarkers that we have described before (39). Western blots of ESC Cx43, ERα, and PR-A and PR-B proteins all showed progressive upregulation after hormone treatment and there was an additive action of Cb during the 5-day incubation period (Fig. 7A). Two-way ANOVA, using laser densitometry data digitized from the corresponding blot bands, indicated positive relationships for days of incubation (F = 8.42, P < 0.001) and treatments (F = 39.01, P < 0.001), but no interactions between time and treatment. Post hoc analyses for all 3 comparisons (control vs H, control vs H + Cb and H vs H + Cb) were significant (P < 0.01, Fig. 7B). A converse, inhibitory effect (F = 24.07, P < 0.001) was noted in experiments analyzing the combined effects of hormones and the DRD2 antagonist, risperidone (Ris), regardless of incubation time. Tukey’s post hoc analyses for all 3 comparison groups (control vs H, control vs H + Ris and H vs H + Ris) were significant (P < 0.01) (Fig. 7C). Like VEGF in Fig. 6C, Cx43, and ERα levels also responded to Cb treatment in the absence of hormones. Maximal stimulation was noted at Cb concentrations of 50 to 75 μM (data not shown) and the time-course is depicted by Western blots in Fig. 7D. After 12 and 48 hours, Cx43 and ERα levels were progressively and significantly higher than control levels (1.8 ± 0.2-fold and 2.6 ± 0.3-fold, ANOVA with Tukey’s post hoc corrections, respectively, P < 0.05). Cabergoline Reverses IL-1β-Mediated Inhibition of ESC Decidualization Our laboratory (39) and other groups (22, 23) have established that IL-1β impedes ESC differentiation in vitro and is a likely contributor to reproductive failure in vivo (44). In the subsequent experiments, we tested the ability of 50 μM Cb to reverse detrimental effects of IL-1β in our ESC decidualization model. Compared with control cells (Fig. 8A), IL-1β exposure for 48 hours induced a characteristic bipolar, fibroblastic phenotype with parallel cell alignment (Fig. 8B) that is associated with loss of MET changes (19). But the spindle-cell phenotype induced by IL-1β was reversed within 48-hour exposure to Cb (Fig. 8D), with cell shape approaching that of ESC treated with Cb alone (Fig. 8C). Quantified cell shape indices and their analysis are shown in Table 2 and document the statistical significance of cell shape changes among the different treatment groups (P < 0.05, ANOVA with Tukey’s tests). Table 2. | ESC treatment (x 48 h) | Cell shape index | (±) SEM | Significant differences | |---|---|---|---| | Control | 0.30 | 0.05 | | | Cabergoline (Cb) | 0.66 | 0.09 | *, ***, | | IL-1β | 0.19 | 0.03 | *, **, **** | | Cb + IL-1β | 0.46 | 0.08 | **, *** | ANOVA P < 0.05 with Tukey’s post hoc tests (* differs from control, ** differs from Cb, *** differs from IL-1β, **** differs from Cb + IL-1β). As observed with the morphological markers of ESC differentiation, inhibitory biochemical effects of IL-1β, as quantified by laser densitometry, also were mitigated by Cb. In ESC derived from 4 independent subjects, in vitro treatment with 0.1 nM IL-1β resulted in 56% ± 8% downregulation of DRD2 (Fig. 9A, n = 4, Student’s t test, P < 0.001), an effect that was paralleled by decreases in other cellular decidualization biomarkers: Cx43 and ERα (69% ± 10% reduction, n = 3, P < 0.001, Fig. 9B). However, co-incubation with Cb completely reversed the inhibitory actions of IL-1β on Cx43, DRD2, ERα, and PR at both the 24-hour and 48-hour time points, resulting in levels above those of untreated controls (Fig. 9C). Due to significant interactions among the time and treatment factors, we could not perform post hoc analysis of 2-way ANOVA data, but 1-way ANOVA indicated that incubation with Cb alone for 24 hours augmented biomarker expression 1.6 ± 0.2-fold above control (n = 3, P < 0.05). The effects appeared less prominent after 48 hours. The pathway responsible for these salutary effects of Cb involves the same MEK-ERK phosphoprotein cascade that we characterized previously (27). IL-1β exposure for 20 minutes induced a 1.8 ± 0.1-fold increase in phospho-ERK, phospho-p90RSK, phospho-p70/85S6K, and phospho-MSK1 (ANOVA, P < 0.05), whereas co-treatment with Cb mostly prevented rapid IL-1β-mediated upregulation of the phosphoprotein cascade (Fig. 9D). We previously reported that expression of these phosphoproteins was correlated with poor ESC decidualization in vitro (31).

E2 and P4, alone, can transform the human endometrium into a vascularized, nutritive, and immune-privileged secretory platform that promotes blastocyst receptivity and early pregnancy (3, 4, 45). We have long suspected that actions of these steroids on the uterine mucosa are mediated via elaboration of local eicosanoids (5), growth factors (6), vitamins (7),and neuroendocrine transmitters (11) synthesized or critically metabolized in situ. One of these neuroendocrine transmitters is dopamine, which derives locally from neurons innervating the endometrial vascular bed. Dopaminergic nerves are characterized by their expression of tyrosine hydroxylase, the rate-limiting enzyme that catalyzes the conversion of L-tyrosine to L-DOPA, which in turn is decarboxylated to dopamine. As we confirmed in Fig. 1, tyrosine hydroxylase-expressing nerves have been identified in human uterus (11, 14), and in other species are located in proximity to the uterine arteries (10). In primates, this is the microanatomic site where the earliest signs of endometrial stromal decidualization are initiated (43). The findings described in this current paper lead us to postulate that the process of neuroangiogenesis (13) recruits catecholaminergic nerves, providing a perivascular source of dopamine that contributes to local stromal cell decidualization via DRD2 activation. A positive-feedback loop appears to be established with hormones and Cb serving to inhibit phosphorylation and activation of the ERK pathway, resulting in an anti-inflammatory environment conducive to ESC differentiation and acquisition of an embryo-receptive phenotype. Although an early study questioned the existence of dopamine receptors in primate endometrium (16), both type 1 (DRD1) and type 2 (DRD2) receptor isoforms were identified later in human decidua (17, 46) and more recently their expression in eutopic endometrium and endometriosis also has been confirmed (18). Our experiments validated strong IHC localization of DRD2 in the endometrial stromal compartment (Fig. 2B) with a subcellular distribution distinct from that of ERα (Fig. 2C). Bioinformatics analysis of single cell RNA sequencing revealed strong expression of DRD2 in mid-secretory endometrium (Fig. 3B). Nevertheless, the function of these receptors remains unknown. Hence, we went on to dynamically test the hypothesis that agonist and antagonist ligands (Cb and risperidone, respectively) of the DRD2 receptor could modulate ESC differentiation in vitro. The findings indicate that DRD2 mRNA and protein are upregulated during the process of hormone-induced ESC decidualization (Figs. 3 and 4) and represent a positive-feedback loop, whereby Cb, through DRD2, further stimulates ESC differentiation. Morphological changes in cell roundness, an MET-related phenomenon (Fig. 5) accompanied biochemical markers (Figs. 6 and 7) that include DRD2 itself along with Cx43, ERα, and PR-A and -B. Risperidone, a DRD2 antagonist, had the converse effect, partly inhibiting time-dependent hormone-induced decidualization (Fig. 7C). However, it should be emphasized that other neurotransmitter receptors, including DRD3, DRD4, and even the serotonin receptors 2A and 2B can bind Cb with high affinity. While expression levels of DRD3 and DRD4 receptors appear to be quite low in ESC, future studies should interrogate the expression and localization of these alternative target proteins in the endometrium. Based on cell shape index determinations, Cb is the most potent ESC differentiating agent that we have evaluated to date. The combination of decidualizing hormones (E2, P4, and cAMP) is the classic stimulus (43), and we have observed that the MEK1/2 kinase inhibitor PD98059 (27) and the PPARβ/δ agonist GW0742 (31), agents with very different mechanisms of action but apparently sharing pathways with Cb, also induce this MET-related morphological phenomenon. It is important to emphasize that 48 to 72 hours is a minimal period to visualize morphological changes in ESC treated with standard decidualizing hormones (19). Relative to a baseline average of 0.34 ± 0.05, the mean Cb-induced cell shape index of 0.63 ± 0.09 within only 48 hours is quite dramatic, as compared with our report of an average cell shape index of 0.83 ± 0.04 after 14 days exposure to E2, P4, and cAMP (19). This observation suggests that the dopamine receptor agonist alone is a strong, rapid, and independent mediator of differentiated phenotype. An important finding in our and others’ investigations focusing on human endometrial receptivity is the recognition that the proinflammatory cytokine IL-1β is implicated in a spectrum of pathophysiological conditions associated with poor pregnancy outcomes (27, 44, 47). Experiments represented in Figs. 8 and 9 reveal that when ESC are incubated with IL-1β, a diminution of DRD2 accumulation results, which is associated with reduced decidual biomarker expression and fibroblastic cell shape. However, co-treatment with Cb completely reversed the inhibitory effects of IL-1β (Fig. 9C), in part by blocking phosphorylation of the MEK-ERK pathway in ESC cultures (Fig. 9D). Although the MEK inhibitor PD had little effect on basal or hormone-induced DRD2 expression (Fig. 4), our previous experiments revealed that IL-1β-mediated inhibition of Cx43, ERα, PR-A, and PR-B can be blocked by PD (39). Cb also appears to reduce IL-1β-induced phosphorylation of critical signaling molecules in the MEK-ERK cascade (Fig. 9D), potentially restoring balance to prevent inflammatory reproductive maladies such as endometriosis (39) and preterm birth (48). A limitation of our study is that it was neither designed nor powered to test the effects of different uterine pathologies on catecholaminergic signaling in the endometrium. Although we excluded endometriosis in the current cohort, participants had leiomyomas or paratubal cysts and some were taking nonsteroidal anti-inflammatory medications at the time of biopsy. While controversy exists based on their anatomic location (49), leiomyomas can exert detrimental pressure and cause inflammatory effects on subjacent endometrial function. To overcome intrinsic, patient-specific variability, we used IL-1β as a potent and reproducible in vitro stimulus to induce inflammation in our model (27). The findings described here support the potential of Cb or other DRD2 agonists as pharmaceutical agents to overcome inflammation-associated decidual dysfunction, but clearly, these in vitro data will need to be corroborated in live animal models. Also, it should be emphasized that chronic and acute inflammation in vivo have multiple reproductive tissue targets, in addition to the endometrium. These include oocyte maturation, fertilization, embryonic development, and other effects on postimplantation uterine health (50). Whether these other targets also might be amenable to reversal with Cb or other dopamine agonists has not been tested to date, but relevant model systems do exist. Murine and nonhuman primate species are expected to be particularly useful in this context for future reproductive toxicity and efficacy analyses. In a retrospective cohort study of women undergoing IVF and zygote intrafallopian transfer, of whom 45 received Cb for prevention of ovarian hyperstimulation syndrome, there was no discernable effect of Cb on embryonic implantation rate (P = 0.10) (51). However, the variance was high and the study was underpowered for this endpoint. Ultimately, large, randomized human clinical trials will be needed to assure that these drugs are safe and effective in early human pregnancy. Acknowledgments The authors thank the operating room and nursing staffs of Wake Forest Baptist Hospital who contributed to the protocol, and we express gratitude for the generous participation of the study participants. We are very appreciative of the assistance of James A. Shelton, MS (University at Buffalo), for his help with the 2-way ANOVA analyses and interpretation. Financial Support: These studies were supported by a Discovery Grant from the Ferring Research Institute (W-000932), an award from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (U01 HD66439) and additional support provided by the Georgia Clinical and Translational Science Alliance of the National Institutes of Health (UL1 TR002378). Author Contributions: All coauthors contributed substantively to the work. Jie Yu, Sarah Berga, Trudy Kohout, Marcel van Duin, and Robert Taylor were responsible for the experimental design. Sarah Berga and Robert Taylor collected the endometrial samples. Jie Yu performed all the cell culture experiments, Qingying Meng did the RNA-Seq bioinformatics analyses and Mingjing Xia ran the Illumina cDNA microarrays. All 7 coauthors analyzed and interpreted the data and prepared the manuscript, Tables, and Figures. Robert Taylor secured funding and wrote the initial drafts of the manuscript, and all 7 coauthors approved the final version of the paper. Glossary Abbreviations - ANOVA analysis of variance - cAMP cyclic adenosine monophosphate - Cb cabergoline - Cx43 connexin 43 - DRD2 type 2 dopamine receptor - E2 17β-estradiol - ELISA enzyme-linked immunosorbent assay - ERα estrogen receptor α - ESC endometrial stromal cells - GAPDH glyceraldehyde 3-phosphate dehydrogenase - H hormones - IHC immunohistochemistry - IL interleukin - IVF in vitro fertilization - MEK-ERK mitogen-activated protein ERK kinase - MET mesenchymal-to-epithelial transformation - P4 progesterone - PD PD98059 (MEK1/2 inhibitor) - PR progesterone receptor - qRT-PCR quantitative reverse-transcriptase polymerase chain reaction - Ris risperidone - SEM standard error of the mean - VEGF vascular endothelial growth factor Additional Information Disclosures: J.Y., S.B., M.X., and R.T. have no conflicts of interest to report. Q.M., T.K., and M.v.D are employees of Ferring Pharmaceuticals. Data Availability The datasets generated and analyzed during the current study will be provided by the corresponding author upon reasonable request.

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[PMC free article] [PubMed] [Google Scholar] Associated Data This section collects any data citations, data availability statements, or supplementary materials included in this article. Data Availability Statement The datasets generated and analyzed during the current study will be provided by the corresponding author upon reasonable request.