Abstract
Endometriosis is an estrogen-dependent, chronic inflammatory disease. Recent studies have shown that vitamin D (VD) is an effective modulator of the immune system and plays an important role in controlling many inflammatory diseases.
The objective of the study was to clarify the in vitro effects of 1,25-dihydroxy vitamin D3 (1,25[OH]2D3) on human endometriotic stromal cells (ESCs) and to determine the serum levels of VD in endometriosis patients.
ESCs were isolated from ovarian endometrioma and cultured with 1,25(OH)2D3. Gene expression of IL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, cytosolic prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, matrix metalloproteinase (MMP)-2, and MMP-9 was examined using quantitative RT-PCR. The production of IL-8 and prostaglandin E2 was measured using an ELISA and an enzyme immunoassay. Viable cell number was assessed using a cell-counting assay, and DNA synthesis was assessed using the bromodeoxyuridine incorporation assay. Apoptosis was assessed using flow cytometry. The expression of inhibitory-κBα protein was detected using Western blotting. The serum levels of 25-hydroxyvitamin D3 and 1,25(OH)2D3 were measured by a RIA.
In vitro studies showed that 1,25(OH)2D3 significantly reduced IL-1β- or TNF-α-induced inflammatory responses, such as IL-8 expression and prostaglandin activity. 1,25(OH)2D3 also reduced viable ESC numbers and DNA synthesis but did not affect apoptosis. MMP-2 and MMP-9 expressions were reduced by 1,25(OH)2D3. 1,25(OH)2D3 inhibited nuclear factor-κB activation. The serum 25-hydroxyvitamin D3 levels were significantly lower in women with severe endometriosis than in the controls and women with mild endometriosis. Serum 1,25(OH)2D3 levels were not different between groups.
VD modulates inflammation and proliferation in endometriotic cells, and a lower VD status is associated with endometriosis. Taken together, VD supplementation could be a novel therapeutic strategy for managing endometriosis.
Endometriosis is an estrogen-dependent, chronic inflammatory disease. It causes pain and infertility and remarkably deteriorates women's health (1). Therapeutic strategies for this disorder are limited to hormonal treatment; consequently, patients who desire to conceive cannot undertake these treatments (2).
Vitamin D (VD) is a pleiotropic molecule and therefore has a broad range of biological activities (3). In addition to its roles in mineralizing the skeleton and in regulating plasma calcium levels, recent studies have shown that VD is an effective modulator of the immune system (4). For example, VD induces dendritic cell tolerance (5) and controls maturation (6), macrophage migration and adhesion (7), cytokine production (8), T lymphocyte proliferation (9), and cytokine production (10).
Recently the antiinflammatory, antiproliferative, and antiinvasive effects of VD have been documented as a result of in vitro studies of synovial fibroblasts in rheumatoid arthritis (11), dendritic cells in psoriasis (12), and cancer cells (13), whereas no such study has been conducted in endometriosis. Regarding the therapeutic effects of VD on endometriosis, a selective VD receptor agonist has been shown to reduce endometriosis development in a mouse model (14); however, the mechanism by which VD controls lesion development is unclear.
The aim of this study was to clarify the in vitro effects of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), a biologically active form of VD, on human endometriotic stromal cells (ESCs). We also determined the serum levels of VD in endometriosis patients.
Materials and Methods
Reagents and materials
Type I collagenase and deoxyribonuclease I were purchased from Wako, and 1,25(OH)2D3 was provided by Teijin Pharma. Antibiotics (a mixture of penicillin, streptomycin, and amphotericin B), IL-1β, and TNF-α were obtained from Sigma. DMEM/F-12 medium, 2.5% trypsin, HEPES, and 0.25% trypsin-EDTA were obtained from Gibco.
Patients and samples
The experimental procedures were approved by the Institutional Review Board of the University of Tokyo. Signed informed consent for the use of tissue or sera was obtained from each participant.
Isolation and culture of human ESCs
Endometriosis tissues for the in vitro study were obtained from patients with ovarian endometriomas (n = 35, women aged 36.7 ± 6.42 y, mean ± SD) who underwent laparoscopies or laparotomies. All patients had not received hormones or GnRH agonists for 3 months or longer before surgery. Endometriosis tissue samples were obtained from the cyst wall of the ovarian endometrioma under sterile conditions and transported to the laboratory on ice in DMEM/F-12.
For isolation and culture, human ESCs were prepared as described previously (15, 16). Endometriosis tissue was minced into small pieces, incubated in DMEM/F-12 with 0.25% type I collagenase, 15 U/mL deoxyribonuclease I, 0.006% trypsin, and 0.02M HEPES for 1–2 hours at 37°C and filtered through nylon cell strainers with apertures of 100 μm and then 70 μm. ESCs were cultured in DMEM/F-12 supplemented with 5% fetal bovine serum and antibiotics. At the first passage, the cells were plated at a density of 2 × 105 cells/well into 6-, 12-, 24-, or 96-well culture plates at 1 × 104 cells/well and then incubated at 37°C in a humidified 5% CO2-95% air environment.
Treatment of ESCs with 1,25(OH)2D3
Each experiment was conducted using samples from different individuals (n = 4–7) because the amount of sample from one individual was insufficient to perform multiple sets of experiments. Unless otherwise indicated, ESCs were cultured in 2%–5% fetal bovine serum medium and treated with 10−7 or 10−6 M 1,25(OH)2D3 for 24 hours. To evaluate the effects of 1,25(OH)2D3 on IL-1β- or TNF-α-induced IL-8 gene expression, the cells were treated with IL-1β (5 ng/mL) or TNF-α (10 ng/mL) in the absence or presence of 1,25(OH)2D3 and incubated for 1, 3, 6, and 24 hours. To evaluate the effects of 1,25(OH)2D3 on IL-1β-induced cyclooxygenase (COX)-2, microsomal prostaglandin (PG) E synthase (mPGES)-1, mPGES-2, cytosolic PGES (cPGES), and 5-hydroxyprostaglandin dehydrogenase (15-PGDH) gene expression, the cells were treated with IL-1β (5 ng/mL) in the absence or presence of 1,25(OH)2D3, and the cells were incubated for 3 hours. This time point was chosen because the authors (17) and others (18) had found that 4 hours is enough time to detect the response of ESCs to IL-1β regarding mRNA expression of PGE2-related enzymes. To evaluate the dose effects of 1,25(OH)2D3 on IL-1β or TNF-α-induced IL-8 and PGE2 secretion, the cells were cultured for 24 hours in the presence or absence of 1,25(OH)2D3 (10−9 to 10−7 M) with IL-1β (5 ng/mL) or TNF-α (10 ng/mL). To evaluate the effects of 1,25(OH)2D3 on inhibitor-κBα (IκBα) protein expression, ESCs were pretreated with 1,25(OH)2D3 for 24 hours and then stimulated with TNF-α (10 ng/mL) for 5 minutes.
RNA extraction, reverse transcription, and real-time PCR
Total RNA was extracted, using an RNeasy minikit (QIAGEN). One microgram of total RNA was reverse transcribed in a 20-μL volume using an RT-PCR kit (TOYOBO). Real-time quantitative PCR was conducted using a LightCycler (Roche Diagnostics) according to the manufacturer's instructions. Expression of each mRNA was normalized for RNA loading for each sample using human glyceraldehyde 3-phosphate dehydrogenase mRNA as the internal standard. Primer information and PCR conditions are listed in Supplemental Table 1. All PCR procedures were followed up with melting curve analysis.
Measurement of IL-8
After treatment, the conditioned media were collected, centrifuged, and stored at −80°C until assayed. IL-8 concentrations were measured using a specific ELISA kit (R&D Systems). The limit of sensitivity of ELISA was 31 pg/mL. There was no cross-reactivity or interference.
Measurement of PGE2
PGE2 concentrations in the media were measured using a specific enzyme immunoassay kit (Cayman Chemical). The limit of sensitivity of the enzyme immunoassay was 7.8 pg/mL. The intra- and interassay coefficients of variation were 8.8% ± 3.2% and 15.6% ± 3.2% (mean ± SEM), respectively.
Viable cell number counting
The viable cell counting assay was performed using the Cell Counting Kit-8 (Dojindo) according to the manufacturer's instructions. This kit measures the activity of dehydrogenase in cells by detecting the yellow formazan dye, which is reduced from tetrazolium by dehydrogenase. Because the activity of dehydrogenase is correlated with the number of living cells, the kit determines the number of living cells. ESCs were treated with or without 1,25(OH)2D3 for 24 hours, after which 10 μL of the cell-counting kit solution was added and cells incubated at 37°C for an additional 2 hours. The resultant color was read at 450 nm in an Epoch microplate spectrophotometer (BioTek).
Bromodeoxyuridine incorporation assay
To evaluate DNA synthesis, the bromodeoxyuridine (BrdU) incorporation assay was performed using the Biotrak cell proliferation ELISA system (GE Healthcare) according to the manufacturer's instructions. Briefly, ESCs were treated with 1,25(OH)2D3 for 24 hours, and 10 μL BrdU solution was added and incubated at 37°C for an additional 2 hours. After removing the culture medium, the cells were fixed and the DNA denatured with the addition of 200 μL/well fixative. Immune complexes (peroxidase labeled anti-BrdU bound to BrdU incorporated in newly synthesized, cellular DNA) were detected by the subsequent substrate reaction, and the resultant color was read at 450 nm using an Epoch microplate spectrophotometer (BioTek).
Assessment of cell death
Apoptosis of ESCs was assessed by double staining for annexin V and propidium iodide and using the Annexin V-EGFP apoptosis detection kit (Abcam) according to the manufacturer's instructions. Briefly, ESCs were detached by 0.25% trypsin-EDTA, washed twice with PBS, and resuspended to 1 × 106 cells/mL in 1× binding buffer. Each sample solution was transferred to a 5-mL culture tube, after which 2 μL of annexin V-fluorescein isothiocyanate and 2 μL of propidium iodide were added. Samples were then incubated for 10 minutes at 4°C in the dark, followed by filtration through a 40-μm nylon mesh (BD Biosciences) to remove cell clumps. After incubation, the samples were analyzed using flow cytometry (FACS Calibur and Cell Quest Pro; BD Biosciences). Annexin V-positive cells were regarded as apoptotic cells.
Western blotting
Cultured cells were homogenized in cell lysis buffer (Cell Signaling Technology). Samples were resolved by 10% SDS-PAGE. Proteins were blotted onto a polyvinylidene difluoride membrane (Amersham Bioscience) and incubated with rabbit anti-IκBα (1:1000; Cell Signaling Technology) as the primary antibody and then antirabbit horseradish peroxidase antibody (1:1000; Santa Cruz Biotechnology, Inc). Immune complexes were visualized us an enhanced chemiluminescence Western blotting system (Amersham).
Measurement of serum levels of 25-hydroxyvitamin D3 (25[OH]VD3) and 1,25(OH)2D3
Serum samples for measuring VD were collected from another set of patients who underwent laparoscopy for treatment of either endometriosis or benign ovarian tumor at the University of Tokyo Hospital (Tokyo, Japan). Seventeen patients with stage 1 or 2 endometriosis (aged 35.4 ± 1.64 y), 22 patients with stage 3 or 4 endometriosis (aged 34.6 ± 1.53 y), and 37 controls who were negative for endometriosis confirmed by laparoscopy (aged 32.8 ± 1.05 y) were enrolled in the study. Endometriosis was staged according to the revised American Society for Reproductive Medicine classification. All patients and controls were negative for fibroids confirmed by laparoscopy. All patients and controls were Japanese residents and Mongoloid. All samples were taken from October to March (autumn to winter). All women were in the proliferative phase of their menstrual cycle. Validation samples (n = 73) were randomly selected from both patients and controls. All sera were collected the day before laparoscopy, and samples were stored at −80°C until the assay.
25(OH)VD3 and 1,25(OH)2D3 were measured by RIA (SRL, Inc). Briefly, after serum samples were mixed with antibody for VD, 125I-labeled VD was added for competitive reactions, and the bound antigens were separated from the unbound ones and measured. Because the RIA has been shown to have problems with specificity, accuracy and sensitivity for the measurement of 25(OH)VD3, and liquid chromatography coupled with tandem mass spectrometry (LS-MS/MS) has provided significant advances and is now considered the gold standard, the results of 25(OH)VD3 concentration measured by RIA were further verified by measuring validation samples using LS-MS/MS (LSI Medience Corp) and a RIA.
Statistical analysis
Data were evaluated using JMP software (version 10.0; SAS Institute Inc). The differences between two samples were calculated using a Student's t test. The differences among multiple samples were calculated using an ANOVA, Tukey's test, and the Mann-Whitney U test. The data are expressed as the mean ± SEM. A value of P < .05 was considered significant.
Results
The effect of 1,25(OH)2D3 on IL-1β- or TNF-α-induced IL-8 mRNA and protein expression
1,25(OH)2D3 significantly reduced IL-1β-induced IL-8 mRNA expression at 6 and 24 hours after the treatments (67.4% ± 9.4% and 72.1% ± 1.7%, respectively, P < .05) (Figure 1A). 1,25(OH)2D3 suppressed the secretion of IL-1β-induced IL-8 protein at a concentration of 10−9 to 10−7 M, with a dose dependency from 10−9 to 10−8 M and a flat response from 10−8 to 10−7 M (Figure 1B). 1,25(OH)2D3 also significantly reduced TNF-α-induced IL-8 mRNA expression at 6 hours after the treatments (80.0 ± 6.0% of controls, P < .05) (Figure 1C). 1,25(OH)2D3 significantly suppressed the secretion of TNF-α-induced IL-8 protein with a flat response from 10−9 to 10−7 M (Figure 1D).
The effect of VD on IL-1β- and TNF-α-induced IL-8 expression.
A, IL-1β (5 ng/mL)-induced IL-8 mRNA expression in ESCs cultured in the absence or presence of 1,25(OH)2D3 (10−7 M) at 1, 3, 6, and 24 hours. B, IL-1β (5 ng/mL)-induced IL-8 protein in supernatant from ESCs cultured in the absence or presence of 1,25(OH)2D3 (10−9 to 10−7 M) for 24 hours. *, P < .001 vs control. C, TNF-α (10 ng/mL)-induced IL-8 mRNA expression in ESCs cultured in the absence or presence of 1,25(OH)2D3 (10−7 M) at 1, 3, 6, and 24 hours. D, TNF-α (10 ng/mL)-induced IL-8 protein in the supernatant from ESCs cultured in the absence or presence of 1,25(OH)2D3 (10−9 to 10−7 M) for 24 hours. *, P < .05 vs control.
The effect of 1,25(OH)2D3 on IL-1β-induced PGE2 secretion, PGE2 synthesis, and degradative enzyme mRNA expression
1,25(OH)2D3 significantly suppressed the secretion of IL-1β-induced PGE2 in a dose-dependent manner (Figure 2A). 1,25(OH)2D3 significantly decreased the expression of IL-1β-induced COX-2, mPGES-1, and mPGES-2 mRNA and increased IL-1β-induced 15-PGDH mRNA expression by ESCs (57.9% ± 12.2%, 74.1% ± 7.4%, 84.1% ± 9.4%, and 224.9% ± 83.4% of controls, respectively, P < .05) (Figure 2, B–D and F). The level of IL-1β-induced cPGES mRNA expression was not changed by 1,25(OH)2D3 (Figure 2E).
The effect of VD on IL-1β-induced PGE2 secretion and PGE2 synthesis and degradative enzyme mRNA expression by ESCs.
A, IL-1β (5 ng/ml) -induced PGE2 levels in supernatant from ESCs cultured in the absence or presence of 1,25(OH)2D3 (10−9 to 10−7 M) for 24 hours. *, P < .05 vs control. B, IL-1β-induced COX-2 mRNA expression. C, IL-1β-induced mPGES-1 mRNA expression. D, IL-1β-induced mPGES-2 mRNA expression. E, IL-1β-induced cPGES mRNA expression. F, IL-1β-induced 15-PGDH mRNA expression. In each instance, ESCs were cultured with IL-1β (5 ng/mL) in the absence or presence of 1,25(OH)2D3 (10−7 M) for 3 hours. *, P < .05 vs control.
Effect of 1,25(OH)2D3 on viable ESC number, DNA synthesis, and apoptosis
1,25(OH)2D3 decreased the number of viable ESCs (90.0% ± 4.1% of controls, P < .05) (Figure 3A). 1,25(OH)2D3 also decreased BrdU incorporation into ESC DNA (73.5% ± 4.6% of controls, P < .05) (Figure 3B). The percentage of apoptotic cells after 1,25(OH)2D3 treatment was not significantly different from that of the controls (Figure 3C).
VD effects on ESC number, DNA synthesis, and apoptosis.
A, Viable ESC numbers using the cell-counting assay. B, BrdU incorporation/DNA synthesis in ESCs. C, Percentage of apoptotic/annexin V-positive ESCs. In each instance, ESCs were treated with 1,25(OH)2D3 (10−6 M) for 24 hours. *, P < .05 vs control.
The effect of 1,25(OH)2D3 on matrix metalloproteinase (MMP)-2 and MMP-9 mRNA expression
Treatment of ESCs with 1,25(OH)2D3 significantly inhibited MMP-2 and MMP-9 mRNA expression (68.4% ± 3.7% and 65.6% ± 5.8% of controls; respectively; P < .05) (Figure 4).
The effect of VD on MMP-2 and MMP-9 mRNA expression by ESCs.
A, MMP-2 mRNA expression. B, MMP-9 mRNA expression. In each instance, ESCs were cultured in the absence or presence of 1,25(OH)2D3 (10−7 M) for 24 hours. *, P < .05 vs control.
The effect of 1,25(OH)2D3 on TNF-α-induced IκBα expression
IκBα protein expression was decreased by TNF-α (34.0% ± 5.4% of controls, P < .05), but this effect was neutralized when ESCs were pretreated with 1,25(OH)2D3 (10−7 M) for 24 hours (87.3% ± 28.6% of controls, P < .05) (Figure 5).
The effect of VD on TNF-α-induced IκBα protein expression by ESCs.
ESCs were treated with TNF-α (10 ng/mL) for 5 minutes after pretreatment with 1,25(OH)2D3 (10−7 M) for 24 hours. Western blotting was performed using antibody against IκBα. A, Data shown are representative of four experiments. B, Bars represent the average ± SEM. *, P < .05 vs control; **, P < .05 vs without 1,25(OH)2D3.
Serum levels of VD
The serum levels of 25(OH)D3 in samples from patients with severe endometriosis (17.2 ± 1.1 ng/mL) were significantly lower than the levels detected in samples from the controls (21.8 ± 1.3 ng/mL, P < .05) and the patients with mild endometriosis (21.5 ± 1.4 ng/mL, P < .01) (Figure 6A). In contrast, the serum 1,25(OH)2D3 levels were not different between the groups (Figure 6B). A strong correlation between the serum levels of 25(OH)D3 measured using RIA and those measured using liquid chromatography coupled with tandem mass spectrometry was found (R2 = 0.6135, P < .05, Figure 6C) in the validation sample, confirming the accuracy of the results using RIA.
![The serum levels of 25-hydroxy vitamin D3 (25[OH]D3 (A) and 1,25(OH)2D3 (B) measured using a RIA.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jcem/101/6/10.1210_jc.2016-1515/2/m_zeg0051625710006.jpeg?Expires=1747852205&Signature=zWq97pOOBAZ6fK5vEo0lGhKj8zdeJlGyKmaxhSupim89e0A3P~YmkN4uRH8tnV3f3r9ksnd6dLKR14UqkgxT~mHaYwQHGAv-nNKDoj8dwG58c4U3NiqXj~rbPoyaItB-Ne3B-fyROXsJ0PxEMWGYBB2c60UhVgEDM8Xq9SkLKepH2aCY7E2dDlp82UoFkxQ~Mc4Mnv04mHKEh~1f2DIzQravD1MIg-GqHtLvr1cCYxwuWOxYQG0fngzqdNlYy8C2kdLl9U4xGaP0v656cy5uP5RIjmdo6XWEqTI-70tarzvpIs4~2RaUoOZI449-uLhP32iNXPHgKQIW34wIZZQ3Qw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
The serum levels of 25-hydroxy vitamin D3 (25[OH]D3 (A) and 1,25(OH)2D3 (B) measured using a RIA.
Serum samples were collected from 37 control women, 17 patients with stage 1 or 2 endometriosis and 22 patients with stage 3 or 4 endometriosis. C, The correlation between the serum concentrations of 25-hydroxyvitamin D3 measured using a RIA and those using LS-MS/MS in validation samples collected from control and patients with endometriosis. A, Serum levels of 25(OH)D3 from stage 3 or 4 endometriosis were significantly lower than levels detected in samples from controls and stage 1 or 2 endometriosis patients. *, P < .05; **, P < .05. B, Serum 1,25(OH)2D3 levels were not different between groups. C, Positive correlation was observed with coefficient of correlation R2 = 0.61345 (P < .05).
Discussion
In this study, we found that 1,25(OH)2D3 significantly reduced IL-1β- or TNF-α-induced inflammatory responses, such as IL-8 mRNA expression, prostaglandin activity, and MMP mRNA expression. Furthermore, 1,25(OH)2D3 regulates the viable ESC number and BrdU incorporation into ESC DNA but had no effect on apoptosis. 1,25(OH)2D3 inhibited NF-κB activation in ESCs. We also demonstrated that the 25(OH)D3 levels were significantly lower in the sera from women with severe endometriosis than in that from the patients with mild endometriosis or controls. These results suggest that VD deficiency is associated with the pathogenesis of endometriosis, and VD supplementation may have therapeutic benefits in endometriosis management.
Treatment of ESCs with 1,25(OH)2D3 significantly reduced inflammatory responses. Inhibitory effects of VD on cytokine production have been demonstrated in various types of cells such as trophoblasts (19), placental cells (20), decidua cells (21), and immortalized myometrial smooth muscle cells (22) in the context of miscarriage, preterm labor, and preeclampsia. Indeed, in support of VD's potential role in the context of reproduction, we demonstrated that 1,25(OH)2D3 significantly reduced IL-1β- or TNF-α-induced IL-8 mRNA expression and production in ESCs, suggesting that VD may exert in vivo regulatory effects on inflammation in endometriosis.
Numerous reports support the assertion that VD regulates the PG pathway in cancer and inflammatory diseases such as asthma. For example, 1,25(OH)2D3 suppresses COX-2 expression in breast (13) and prostate (23) cancer cells and stimulates 15-PGDH in breast cancer cells (13). PG-inhibitory effects have also been shown in lung fibroblasts; for instance, 1,25(OH)2D3 significantly reduced PGE2 production and IL-1β-induced mPGES-1 and stimulated 15-PGDH (24). Given that the PG pathway plays important roles in the pathophysiology of endometriosis (25), we tested the effect of VD on ESCs. Similar to other cell type responses, 1,25(OH)2D3 significantly reduced PGE2 production by ESCs. This may be owing to the reduction of PG-synthetic enzymes such as COX-2, mPGES-1, and mPGES-2 and the increase of the PG-degradative enzyme 15-PGDH and may reveal a mechanism by which VD affects endometriosis development.
We found that 1,25(OH)2D3 significantly decreased viable ESC numbers as a result of reduced DNA synthesis rather than the induction of apoptosis. Previous reports have indicated that VD is proapoptotic in normal (26) or cancerous (13) cells, but this was not the case in ESCs. Endometriosis is resistant to apoptosis (27), and this may at least partially explain why VD did not induce apoptosis in ESCs.
VD can regulate MMP expression in endometrial cancer (28) and uterine fibroid cells (29). High MMP-2 and MMP-9 levels in serum or tissue (30) have been associated with the pathogenesis of endometriosis. Interestingly, the expression of MMP-9 in an endometriosis murine model was reduced by the administration of VD (31). In consideration of these findings, we examined the effect of 1,25(OH)2D3 on MMP-2 and MMP-9 expression in ESCs. Given that 1,25(OH)2D3 significantly reduced MMP mRNA expression, it is possible that VD exerts an antiinvasive effect by inhibiting MMP production. Further studies such as invasive assays or xenograft studies could be used to explore this mechanism.
We further demonstrated that 1,25(OH)2D3 reduced activation of the NFκB pathway in ESCs by preserving IκBα. Because NFκB mediates cytokine or prostaglandin production (32), MMP expression (33), and cell proliferation (34), our findings reveal the possible mechanism for the antiinflammatory, antiproliferative, and anti-MMP effects of 1,25(OH)2D3 reported in earlier studies. VD-induced inhibition of the NFκB pathway has also been reported in other cell types in various pathological conditions, such as uterine myometrial cells in preterm labor (35), peripheral blood mononuclear cells in Crohn's disease (36), and adipocytes in obesity (37); consequently, VD's therapeutic potential has been proposed.
There were several limitations in the current in vitro study. First, the ESCs were only from endometriomas and therefore cannot be considered as representative of all endometriosis. Second, assays of cell secretions such as cytokines are possibly affected by cell viability, although the observed differences in secretions were greater than those in viability.
Our findings, indicating an association between low serum levels of 25(OH)D3 and the severity of endometriosis, are consistent with a previous report (38); however, other groups have reported contrary results, ie, higher 25(OH)D3 levels with endometriosis (39) or no difference from that of healthy controls (40, 41). These discrepancies may be due to the variations in race and country of residence in these studies. Our study clarified, however, that serum 25(OH)D3 is reduced in Mongoloid and Japanese patients diagnosed with severe endometriosis and residing in Japan. It is also possible that seasonal influences (42), menstrual cycle, and disease stage influence 25(OH)D3 levels. Our study minimized these influences by sampling in autumn and winter during the proliferative phase of the menstrual cycle, and comparisons were made according to disease severity. Taken together, our results indicate that VD deficiency is associated with the progression of endometriosis.
In contrast to 25(OH)D3, the levels of 1,25(OH)2D3 did not differ between the groups. 1,25(OH)2D3 is the biologically active form of VD and is therefore the relevant form for use in in vitro studies. Circulating levels, however, do not reflect the systemic VD status because of its short half-life (43). Therefore, a negligible difference in 1,25(OH)2D3 levels between groups does not refute the association between VD status and endometriosis.
Having shown the association between low serum VD status and severe endometriosis and the antiinflammatory, antiproliferative, and antiinvasive properties of VD in ESCs, we propose that VD deficiency may promote the progression of endometriosis; therefore, VD supplementation may be used to manage the disease. Recently VD supplementation trials have indicated that the vitamin is an effective therapeutic for many medical disorders, including Crohn's disease (44). Given that VD does not affect ovulation, VD supplementation may be an ideal therapy for those who wish to conceive. Clinical studies to evaluate the efficacy of VD on endometriosis symptoms and lesions are warranted in the future.
In summary, this study demonstrated that VD may prevent disease progression and that lower VD status was associated with endometriosis; therefore, VD supplements should be explored as a novel therapeutic strategy for managing the disease.
Acknowledgments
We thank the medical colleagues in the University of Tokyo Hospital for collecting clinical samples and Dr Kate Hale for editing the manuscript.
This work was supported by grants from the Ministry of Health, Labor and Welfare, the Ministry of Education.
Disclosure Summary: The authors have nothing to disclose.
Abbreviations
- BrdU
bromodeoxyuridine
- COX
cyclooxygenase
- cPGES
cytosolic PGES
- ESC
endometriotic stromal cell
- IκBα
inhibitor-κBα
- LS-MS/MS
liquid chromatography coupled with tandem mass spectrometry
- MMP
matrix metalloproteinase
- mPGES
microsomal PGE synthase
- 1,25(OH)2D3
1,25-dihydroxyvitamin D3
- 25[OH]VD3
25-hydroxyvitamin D3
- PG
prostaglandin
- 15-PGDH
15-hydroxyprostaglandin dehydrogenase
- VD
vitamin D.
