Elevated levels of adrenomedullin in eutopic endometrium and plasma from women with endometriosis

Brooke C. Matson, Matson BC, Kelsey E. Quinn, Lessey BA, Bruce A. Lessey, Steven L. Young, Kathleen M. Caron
Fertility and sterility · 2018 · vol. 109(6) , pp. 1072–1078 · doi:10.1016/j.fertnstert.2018.02.004 · PMID:29871794 · PMC6015786 · W2807703887
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Adrenomedullin levels were elevated in the eutopic endometrium and plasma of women with endometriosis, suggesting MR-proADM may serve as a biomarker.

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This paper investigated whether adrenomedullin (Adm/AM) is regulated by the IL-6–STAT3 pathway in uterine tissue and whether a stable circulating precursor, mid-regional pro-adrenomedullin (MR-proADM), could serve as a biomarker for endometriosis. Using Ishikawa endometrial cells treated with IL-6, the authors found IL-6 caused dose-dependent STAT3 phosphorylation and increased Adm gene expression, and they observed higher AM immunostaining in multiple compartments (luminal epithelium, glandular epithelium, and stroma) of eutopic endometrium from women with endometriosis; they report that MR-proADM plasma levels were elevated in women with endometriosis, did not vary with menstrual cycle phase or disease stage, and persisted after surgical resection, with no association to short-term fertility outcome. Limitations explicitly include reliance on comparisons using an unpaired t-test/ANOVA approach without correction details, and MR-proADM remained unchanged by stage and surgery. This paper is centrally about endometriosis—specifically testing whether the IL-6/STAT3–adrenomedullin axis is upregulated in eutopic endometrium and whether MR-proADM is elevated as a biomarker in plasma.

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Abstract

ObjectiveTo test adrenomedullin (Adm, ADM) as a downstream target of signal transducer and activator of transcription 3 (STAT3) in endometrial cells and to test midregional proadrenomedullin (MR-proADM) as a biomarker of endometriosis.DesignCross-sectional analysis of Adm expression in eutopic endometrium and of MR-proADM in plasma from women with and without endometriosis; and prospective study of MR-proADM levels in women with endometriosis undergoing surgical resection of ectopic lesions.SettingAcademic medical centers.Patient(s)Fifteen patients with endometriosis and 11 healthy control subjects who donated eutopic endometrial biopsies; and 28 patients with endometriosis and 19 healthy control subjects who donated plasma for MR-proADM analysis.Intervention(s)None.Main outcome measure(s)Adm mRNA levels according to quantitative real-time polymerase chain reaction after activation of STAT3 by interleukin-6 (IL-6) in Ishikawa cells; immunohistochemistry for ADM in eutopic endometrial biopsies from women with endometriosis compared with healthy donors; and MR-proADM levels measured by commercial immunoassay in plasma from healthy women and women with endometriosis who subsequently underwent surgical resection of ectopic lesions.Result(s)Activation of STAT3 by IL-6 up-regulated Adm mRNA expression in Ishikawa cells. ADM protein levels were elevated in the eutopic endometrium of women with endometriosis. MR-proADM concentrations were higher in women with endometriosis but were not correlated with disease stage, corrected by surgery, or predictive of fertility outcome.Conclusion(s)MR-proADM may be able to serve as a biomarker of endometriosis, but it is unknown whether elevated MR-proADM levels are secondary to the estrogenic and inflammatory properties of endometriosis or an inciting pathogenic factor.
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Intro

Endometriosis is a common gynecological disease characterized by the presence of endometrial tissue outside of the eutopic endometrium, commonly in the peritoneum of the pelvis and in pelvic organs, often causing scarring and pain ( 1 , 2 ). Although endometriosis is also strongly associated with subfertility and infertility, the mechanisms underlying fertility problems in women with stage 1 to 2 disease is a subject of debate in the field ( 3 , 4 ). The non-specific nature of endometriosis symptoms makes the disease difficult to diagnose, and confident diagnosis usually requires visualization of ectopic lesions at surgical exploration. Endometriosis is equally difficult to treat; hormonal therapies and surgical excision of ectopic lesions are the mainstays of treatment but are not always effective ( 5 , 6 ). These diagnostic and therapeutic challenges have fueled interest in identifying biomarkers and signaling pathways associated with endometriosis ( 7 ). Recently, Kim et al. demonstrated aberrant activation of signal transducer and activator of transcription 3 (STAT3) in the eutopic endometrium of women with endometriosis ( 8 ). Other studies consistently point to endometriosis-associated factors that affect STAT3 activation: the cytokine interleukin 6 (IL-6), which activates STAT3 through the IL-6 receptor, is elevated in the peritoneal fluid of women with endometriosis ( 9 – 11 ); miR120, which targets STAT3, is elevated in endometriotic cyst stromal cells ( 12 ); and protein inhibitor of activated STAT3 (PIAS3) and dual-specificity phosphatase-2 (DUSP2), negative regulators of STAT3, are downregulated in endometriosis ( 13 , 14 ). Kim et al. also found hypoxia inducible factor 1A (HIF1A), which is stabilized by STAT3, to be elevated in the eutopic endometrium of women with endometriosis. Taken together, these data strongly implicate the IL-6-STAT3-HIF1A pathway in the pathophysiology of endometriosis ( 8 ). Both STAT3 and HIF1A have been previously identified as regulators of the versatile peptide hormone adrenomedullin ( Adm , AM) ( 15 – 18 ). AM is expressed in the female reproductive system and has been associated with female reproductive physiology, including embryo implantation and placentation, and in pathophysiology, including subfertility and complications of pregnancy like preeclampsia ( 19 , 20 ). In endometriosis, AM is higher in intrafollicular fluid and negatively associated with oocyte maturity and embryo quality in women with endometriosis, underscoring a potential link between AM, endometriosis, and fertility ( 21 ). Collectively, these data imply that AM may be able to serve as a biomarker of endometriosis. Mid-regional pro-adrenomedullin (MR-proADM) is a byproduct of post-translational processing of preproAM peptide and is a stabler analyte than the mature AM peptide ( 22 ). In the past decade, many groups have found prognostic value for MR-proADM plasma concentrations as a biomarker of heart failure ( 23 ), community-acquired pneumonia ( 24 ), and sepsis ( 25 ), among other diseases. In reproduction, MR-proADM has been tested as a biomarker of gestational diabetes and preeclampsia ( 26 , 27 ). Here, we test the hypothesis that MR-proADM, as a surrogate for AM potentially downstream of the IL-6-STAT3 axis, can serve as a biomarker of endometriosis.

Results

Given elevated levels of IL-6 and phosphorylated STAT3 (pSTAT3) in peritoneal fluid and eutopic endometrium, respectively, of women with endometriosis ( 9 – 11 ), and prior evidence for STAT3 regulation of Adm expression ( 17 , 18 ), we tested whether IL-6 could induce Adm expression in an endometrial cell line, Ishikawa cells. First, we treated Ishikawa cells with increasing doses of IL-6 to confirm that IL-6 induced phosphorylation of STAT3 in Ishikawa cells. Indeed, we observed an IL-6 dose-dependent increase in pSTAT3 by western blot ( Fig. 1A ). We then assessed whether IL-6-mediated phosphorylation of STAT3 induced Adm expression, finding that treating Ishikawa cells with 100 ng/mL IL-6 for 15 minutes upregulated Adm gene expression approximately 1.4-fold by qRT-PCR ( Fig. 1B ). Considering the elevation of pSTAT3 in endometriotic endometrium coupled with our in vitro evidence for STAT3-mediated upregulation of Adm expression in endometrial cells, we asked whether AM is also upregulated in the endometrium of women with endometriosis. Indeed, we found that AM staining was significantly greater in luminal epithelium, glandular epithelium, and stroma of eutopic endometrium from women with endometriosis compared to healthy controls ( Fig. 2 ). We then asked whether circulating plasma levels of MR-proADM, a stable precursor to the mature AM peptide, are elevated in women with endometriosis. The clinical characteristics of healthy controls and women with endometriosis who donated plasma for MR-proADM analysis are displayed in Table 1 . First, we confirmed that MR-proADM levels are stable across all phases of the menstrual cycle in healthy women ( Fig. 3A ). MR-proADM levels averaged approximately 0.35 nmol/L, which is nearly equivalent to the mean MR-proADM concentration of 0.33 nmol/L across the general population ( 22 ). Subsequently, and consistent with higher levels of AM in eutopic endometrium of women with endometriosis, we found that circulating plasma levels of MR-proADM were higher in women with endometriosis compared to healthy controls ( Fig. 3B ). However, MR-proADM levels did not vary by stage of disease ( Fig. 3C ). We then asked whether the elevated levels of MR-proADM were corrected by surgical resection of ectopic endometrial lesions. Analyzing MR-proADM concentrations immediately after surgery and three months after surgery, we determined that elevated MR-proADM levels persisted through the post-surgical period ( Fig. 3D ). Finally, we assessed whether MR-proADM concentrations before, after, and three months after surgery correlated with whether participants were able to become pregnant. We did not find any difference in MR-proADM levels between women with endometriosis who became pregnant and those who did not ( Fig. 3E and F ).

Discussion

In this study, we have presented evidence for Adm as a STAT3 target in the uterus and investigated MR-proADM as a potential biomarker of endometriosis. Prior evidence for a STAT3- Adm axis is two-fold: first, pSTAT3 and Adm levels are positively correlated in breast cancer ( 18 ); and second, activation of STAT3 by oncostatin M promotes Adm transcription in astroglioma cells ( 17 ). However, to our knowledge, this is the first direct evidence for a STAT3- Adm axis in the uterus. This finding is not surprising, as pSTAT3 and AM are co-localized in the uterus during early pregnancy ( 28 , 29 ). Furthermore, reduction of STAT3 or AM in the mouse uterus leads to problems with implantation, potentially due to problems with endometrial receptivity ( 28 – 31 ). It will be the subject of future studies to determine whether STAT3 is directly binding active sites in the Adm promoter in uterine cells or whether the STAT3-mediated stabilization of HIF1A induces Adm transcription. Furthermore, it will be pertinent to determine if this interaction is critical for reproductive physiology and pathophysiology, specifically in humans and in the context of endometriosis. We then asked whether AM, like pSTAT3, was upregulated in the eutopic endometrium of women with endometriosis, finding more AM in both epithelial and stromal compartments of the uterus. The increased expression is consistent with previously demonstrated elevated pSTAT3 and HIF1A abundance in the eutopic endometrium of women with endometriosis, echoing the possibility of an indirect relationship between STAT3 and Adm via HIF1A ( 8 ). However, the expression of AM in ectopic lesions remains unknown. Because MR-proADM is a stable surrogate for AM peptide in circulating plasma, we compared levels of MR-proADM in women with and without endometriosis. This is the first study, to our knowledge, to examine levels of MR-proADM across the menstrual cycle. As the MR-proADM assay was developed just over a decade ago, reference intervals in different physiological conditions and disease states are just emerging ( 32 ). Here, we contribute evidence for stable levels of MR-proADM across all stages of the menstrual cycle in healthy women. Comparison of MR-proADM concentrations in women with and without endometriosis led to our primary finding of elevated MR-proADM in women with endometriosis. We posit several explanations for the finding: First, endometriosis is well-understood to be an estrogenic disease, and Adm is transcriptionally regulated by estrogen ( 33 , 34 ). Second, endometriosis is an inflammatory condition with consequences for fertility, and AM is an anti-inflammatory peptide commonly upregulated in response to inflammation ( 35 , 36 ). Endometriosis is also associated with an elevated risk of epithelial ovarian cancer, and AM has been shown to promoter angiogenesis in this subtype of ovarian cancer ( 37 – 39 ). Therefore, our finding of elevated MR-proADM in women with endometriosis is consistent with associations common to both endometriosis and AM. However, MR-proADM levels were not correlated with disease stage or corrected by surgical resection of ectopic lesions. Endometriosis staging determined primarily by the location and extent of ectopic lesions, and surgical resection aims to remove these lesions. Therefore, if ectopic lesions were the primary source of circulating MR-proADM, we would expect MR-proADM levels to be positively correlated with disease stage and to be corrected by surgery. While the expression of AM in ectopic lesions is not examined in this study, together, these data suggest that ectopic lesions are not the primary source of circulating MR-proADM. MR-proADM levels were not predictive of fertility outcome in women with endometriosis, which is associated with subfertility or infertility, in women who underwent surgery. In our study, therefore, MR-proADM may not demonstrate a prognostic value in endometriosis that is equivalent to that observed by other groups in cardiovascular and pulmonary diseases. However, studies by our group and others demonstrate that levels of both maternal- and fetal-derived AM are important for fertility and pregnancy ( 19 , 20 ). It appears that AM levels are carefully titrated in normal physiology; for example, while Adm −/− mice are embryonic lethal, Adm hi/hi mice, which overexpress AM, develop hyperplastic hearts during development ( 40 , 41 ). Therefore, it is becoming clear that concentrations of AM at either extreme can be detrimental to normal physiology. In summary, we present evidence for a STAT3- Adm interaction in vivo ; for upregulation of AM in the eutopic endometrium of women with endometriosis; and for higher circulating levels of MR-proADM in endometriosis. Whether elevated MR-proADM in endometriosis is secondary to the estrogenic and inflammatory properties of endometriosis or an inciting pathogenic factor will be the subject of future study.

Materials|Methods

The study was approved by the Institutional Review Boards of Greenville Health System and the University of North Carolina at Chapel Hill. Informed consent was obtained from all study participants, who were between the ages of 18 and 45 and had not used hormonal therapies or an intrauterine device in the three months preceding biopsy or plasma collection. Eutopic endometrial biopsies were collected from healthy donor women and women with endometriosis in both proliferative and secretory phases at the time of surgery at Greenville Health System and the University of North Carolina. Plasma samples for analysis of MR-proADM concentrations were collected from healthy women and from women with endometriosis in both proliferative and secretory phases at Greenville Health System and the University of North Carolina. Patients who wished to conceive were followed expectantly after surgery for up to 6 months and pregnancies recorded. Pregnancy was defined as a visible gestational sac on ultrasound with cardiac activity and referral for obstetrical care. The clinical characteristics of women from whom plasma was collected is displayed in Table 1 . MR-proADM concentrations were measured in undiluted plasma using a commercial assay (BRAHMS MR-proADM KRYPTOR) by Phadia Immunology Reference Laboratory. Five micron sections of paraffin-embedded endometrial biopsies were deparaffinized and hydrated. Following antigen retrieval in 10 mM citric acid/0.05% Tween 20, pH 6.0, endogenous peroxidase activity was quenched with 3% hydrogen peroxide in phosphate buffered saline (PBS). Tissues were permeabilized with PBS/0.1% Triton X-100 (PBST) and then blocked in 10% normal goat serum/1% bovine serum albumin in PBST. Tissues were incubated in anti-adrenomedullin primary antibody (1:200, Abcam ab69117) in block overnight at room temperature. The following day, slides were washed and incubated in biotinylated goat anti-rabbit (1:250, Jackson ImmunoResearch) for one hour. Avidin-biotin complexes (VECTASTAIN Elite ABC Kit, Vector Laboratories) were added to tissues for 30 minutes, and then diaminobenzidine (DAB Peroxidase (HRP) Substrate Kit, Vector Laboratories) was added for two minutes. Slides were rinsed with tap water, counterstained with hematoxylin (Vector Laboratories) for 20 seconds, and then rinsed with tap water again. Tissues were dehydrated and then coverslipped using DPX mountant (VWR). Slides were imaged on a Zeiss AxioImager with ProgRes CapturePro software (Jenoptik). Staining intensity was determined by a blinded observer (KEQ) and graded on a scale of 0 (no staining) to 4 (strong staining). Ishikawa cells were cultured in DMEM/F12 (Gibco) + 10% fetal bovine serum (FBS) + 1x penicillin/streptomycin (Gibco) in a 37°C incubator containing 5% CO 2 . For western blot analysis, Ishikawa cells were grown to confluency in 10 cm dishes and were treated with a vehicle control or 1, 10, or 100 ng/mL human IL-6 (R&D Systems) for 15 minutes. Cells were lysed in PBS + 10 nM NaF + 2 mM Na 3 (VO 4 ) + 2mM PMSF + protease inhibitor cocktail, and protein concentration in the lysates was determined using a BCA Protein Assay Kit (Pierce). Twenty micrograms of protein per sample were loaded on a SDS-PAGE gel (Bio-Rad) and then transferred to a nitrocellulose membrane (GE Healthcare Life Sciences). Membranes were blocked in either 5% bovine serum albumin (Fisher Scientific) or 5% nonfat dry milk for one hour at room temperature and then incubated overnight at 4° in primary antibodies: phospho-STAT3 (1:2,000 in 5% BSA, Cell Signaling) or STAT3 (1:2,000 in 5% milk, Cell Signaling). Blots were washed three times in tris-buffered saline/0.1% Tween 20 and then incubated for two hours at room temperature in secondary antibodies: DyLight 680 goat anti-mouse (1:15,000, Thermo Scientific) and DyLight 680 goat anti-rabbit (1:15,000 Thermo Scientific). Membranes were then imaged on an Odyssey CLx (LI-COR). For gene expression analysis, Ishikawa cells were grown to near confluency in 10 cm dishes and then serum starved overnight in serum-free media. Cells were treated with 100 ng/mL IL-6 (R&D Systems) for 1 hour. RNA was collected and isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. Complementary DNA was synthesized from 2000 ng of DNase-treated RNA using M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR was performed using a human Adm Assay on Demand (Applied Biosystems, Hs00969450_g1) and human GAPDH primers and probe (Applied Biosystems, 4310884E) on a StepOne Plus (Applied Biosystems). qRT-PCR data was analyzed using the 2 −ΔΔCt method. All statistical analyses were performed in Prism 5 (GraphPad Software, Inc.). Adm gene expression in vehicle- and IL-6 treated Ishikawa cells; AM staining intensity in endometrial biopsies from healthy women and women with endometriosis; and MR-proADM concentrations in plasma from healthy women and women with endometriosis were compared by unpaired t-test. MR-proADM concentrations by menstrual cycle phase; stage of disease; surgical status; and fertility outcome were compared by one-way ANOVA. Data was considered statistically significant if p<0.05.

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endometriosis

MeSH descriptors

Adrenomedullin Endometriosis Endometrium Uterine Diseases Adrenomedullin Adrenomedullin Adrenomedullin Adult Biomarkers Biomarkers Biomarkers Biomarkers Blood Chemical Analysis Case-Control Studies Cells, Cultured Cross-Sectional Studies Endometriosis Endometriosis Endometriosis Endometriosis

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