The effect of progesterone on genes involved in preterm labor

Accepted Manuscript Title: The effect of progesterone on genes involved in preterm labor Author: Hitomi Okabe Shintaro Makino Kiyoko Kato Kikumi Matsuoka Hiroyuki Seki Satoru Takeda PII: DOI: Reference:

S0165-0378(14)00038-2 http://dx.doi.org/doi:10.1016/j.jri.2014.03.008 JRI 2254

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Journal

Received date: Revised date: Accepted date:

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Please cite this article as: Okabe, H., Makino, S., Kato, K., Matsuoka, K., Seki, H., Takeda, S.,The effect of progesterone on genes involved in preterm labor, Journal of Reproductive Immunology (2014), http://dx.doi.org/10.1016/j.jri.2014.03.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Manuscript

The effect of progesterone on genes involved in preterm labor

Hitomi Okabea, Shintaro Makinoa*, Kiyoko Katoa, Kikumi Matsuokab, Hiroyuki Sekib,

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Satoru Takedaa 5

of Obstetrics and Gynecology, Juntendo University Faculty of Medicine,

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aDepartment

bCenter

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Tokyo, Japan

for Maternal, Fetal and Neonatal Medicine, Saitama Medical Center, Saitama,

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Japan

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*Corresponding author: Shintaro Makino M.D., PhD.

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Department of Obstetrics and Gynecology, Juntendo University Faculty of Medicine,

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2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Tel: +81-3-5802-1100; Fax: +81-3-5689-7460; E-mail address: [email protected] 15

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Abstract The decidua is known to be a major source of intrauterine PGF2α during late gestation

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and labor, and inflammatory cytokines, including IL-1β, IL-6, and IL-8, are elevated in spontaneous preterm deliveries. In the present study, to elucidate how progesterone blocks the pathways associated with preterm birth, we determined the effects of P4 on

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the expression of PTGS-2 and PTGFR mRNA in human decidua fibroblast cells, as well

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as the genes, using microarray analysis. Senescence was induced in primary cultured

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human decidual cells treated with IL-1β. The IL-1β treatment implicated by microarray analysis increased gene expression levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8. In contrast, P4 + IL-1β decreased the expression levels of all of these genes in

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comparison to treatment with IL-1β alone (p<0.05). IL-1β also increased the proportion of SA-β-gal-positive cells. Treatment with IL-1β also increased the p21 protein level in comparison to cells treated either with the vehicle or P4. Neither the p21 protein level nor the number of SA-β-gal-positive cells was increased in normal endometrial 30

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glandular cells by IL-1β (p<0.05). Our studies demonstrated that P4 changes the level of gene expression in a manner that favors an anti-inflammatory milieu. Because IL-8 appears to be the cytokine whose expression is most significantly modulated by P4, further studies evaluating IL-8 as a therapeutic target are needed.

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Keywords

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IL-1β, IL-8, P4, NFκ-B p65, p21

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Abbreviations CX-43, connexin43 FBS, fetal bovine serum FDA, Food and Drug Administration

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HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid

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IGFBP-1, insulin-like growth factor binding protein-1 IL, interleukin (e.g., IL-1) 45

NFκ-B, nuclear factor-kappa B

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OTR, oxytocin receptor P4, progesterone PRL, prolactin receptor 50

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PGF2α, prostaglandin F2α

PROM, premature rupture of membranes PTGFR, prostaglandin F2α receptor

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PTGS-2, prostaglandin-endoperoxide synthase2 / also known as COX-2 = cyclooxygenase-2

17OHP-C, 17 alpha-hydroxyprogesterone caproate 55

RT-PCR, reverse transcriptase polymerase chain reaction SA-β-gal, senescence associated-beta-galactosidase

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VP, vaginal progesterone

1. Introduction 60

Spontaneous preterm labor and delivery occur after premature labor with intact membranes, prelabor rupture of membranes, and cervical insufficiency (Romero et al., 2006a, 2006b). Intra-amniotic infection accounts for 25%, and it activates all 3 Page 3 of 40

components of the common pathway of parturition, not only cervical ripening, but also evidence of myometrial activation and membrane/decidual activation (Tromp et al., 2004; Belt et al., 1999; Romero et al., 2006a, 2006b).

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The prostaglandin F2α receptor (PTGFR) and prostaglandin-endoperoxide synthase-2

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(PTGS-2) are related to the induction of labor. Fuchs et al. (1982) reported that the

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decidua is the major source of intrauterine PGF2α during late gestation and labor. In general, the expression of myometrial PTGFR mRNA decreases progressively throughout gestation in the absence of labor. Moreover, the decidua produces large

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concentrations of prostaglandin F2α (Willman & Collins, 1976) and oxytocin (Chibbar et al., 1993). We have demonstrated that the decidua expresses PTGS-2 mRNA and protein,

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the oxytocin receptor (OTR), connexin43 (CX-43), and PTGFR; however, PTGFR was the only gene/protein in which there was an additional increase in expression at late 75

gestation within this tissue (Makino et al., 2007).

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Progesterone (P4) was discovered as a hormone produced by the corpus luteum, essential for pregnancy maintenance (Csapo et al., 1971, 1972; Lucovnik et al., 2011). In the first trimester, P4 produced by the corpus luteum is critical to the maintenance of early pregnancy until the placenta assumes this function at 7–9 weeks’ gestation. In 80

2011, the US Food and Drug Administration (FDA) approved the use of 17 4 Page 4 of 40

alpha-hydroxyprogesterone caproate (17OHP-C) supplementation during pregnancy to reduce the risk of recurrent preterm birth in women with a history of at least one prior

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spontaneous preterm delivery (Meis et al., 2003). Exogenous administration of 17P is

Connors, 2004; Meis et al., 2005).

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now widely used for the prevention for premature delivery (Meis et al., 2003; Meis &

Vaginal progesterone (VP), natural progesterone (not the synthetic progestin), and

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17OHP-C have been shown to prevent preterm birth (Hassan et al., 2000; Romero &

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Stanczyk, 2013; Kuon et al., 2010). These findings suggest that neither VP nor 17OHP-C has a significant effect on pathways involved in uterine activity or cervical remodeling in abnormal murine pregnancies (Koumans et al., 2012).

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Thus, contrary to the expectation that progesterone blocks pathways associated with structural remodeling of the ante partum cervix, administration of VP has only been noted to increase the expression of the antimicrobial protein, defensin1 (Nold et al.,

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2013). 95

For women with a short cervix, vaginal progesterone reduced the rate of preterm labor (Romero et al., 2013). In women with a short cervix and a prior history of preterm birth, two strategies have been shown to be equally as effective:-vaginal progesterone and cervical cerclage (Conde-Agudelo et al., 2013). However, the mechanism underlying the

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effect of P4 on the prevention of preterm labor is still unknown.

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p53 may also play a role in maintaining uterine quiescence in mice. Observations of

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senescence-associated restriction of uterine growth and preterm birth following the

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conditional deletion of uterine p53 in mice have revealed the critical role of p53 in

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uterine biology and parturition involving the p21/Akt/PTGS-2 pathway (Hirota et al., 2010). Senescence-associated growth restriction with increased levels of p21 in decidual cells is also likely involved in the pathophysiology of preterm labor (Hirota et al., 2010).

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The purpose of the studies reported herein is to examine the mechanisms whereby

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progesterone may prevent preterm delivery. We studied the effect of P4 on the expression of PTGS-2 and PTGFR mRNA in decidual cells using microarray

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experiments as well as studying senescence in primary cultured human decidual fibroblast cells.

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2. Materials and Methods

2.1. Tissue collection and processing

The primary cell cultures derived from decidual fibroblast cells (decidual cells) were 115

obtained following elective cesarean section from 15 patients at 37–38 weeks’ gestation;

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the cells were isolated and cultured as previously described (Keelan et al., 1997). All patients provided informed consent at the Department of Obstetrics and Gynecology,

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Juntendo University Faculty of Medicine and Saitama Medical Center and this protocol

Samples were obtained from pregnant women who met the following inclusion criteria:

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was approved by the institutional ethics committee.

median age (range) of 34.5 (20–40) years and single pregnancy. Exclusion criteria

such

as

diabetes,

hypertension

or

autoimmune

disease;

and

3)

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conditions

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included: 1) use of tobacco or alcohol during the pregnancy; 2) pre-existing clinical

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pregnancy-related complications such as induced hypertension, intrauterine growth restriction, and bleeding (Hanna et al., 2006) ( Table 1).

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Briefly, fetal membranes were washed three times with RPMI 1640 (Sigma Aldrich, St. Louis, MO, USA) to remove adherent blood vessels/clots. The decidua was scraped off the underlying chorioamnion with a glass slide, washed in RPMI 1640 medium, and

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minced with a scalpel. After centrifugation at 1200 × g for 5 min at 24°C, the pellet was 130

resuspended in RPMI 1640 and incubated at 37°C for 10 min. After standing for 5 min to settle, the supernatant portion was collected and centrifuged at 1200 × g for 5 min. This step resulted in the purification of decidual cells. The attached cells were cultured in the flask in RPMI 1640 (supplemented with 10% FBS, penicillin, and streptomycin) 7 Page 7 of 40

until the cells reached confluency for 14 days at a density between 5×105 and 1×106 135

cells/well in plastic plates (25 mm in diameter). The cultures were maintained at 37°C

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in a humidified atmosphere consisting of 95% air and 5% CO2.

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The medium was then replaced with serum-free RPMI 1640 medium containing IL-1β (5

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ng/ml; R＆D Systems, Minneapolis, MN, USA. (Friebe-Hoffmann et al., 2007; Keelan et al., 1997) and crystalline P4 dissolved in ethanol (P4, 1×10-6 M; Sigma Aldrich) (Chanrachakul et al., 2005; Ishihara et al., 1995).

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To confirm that decidual cells were present in the cultures, the levels of prolactin

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receptor (PRL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and insulin-like growth factor binding protein-1 IGFBP-1; (R＆D Systems) were investigated in the cell

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lysate using Western blot analysis (Richards et al., 1995; data not shown). The levels of PRL and IGFBP-1 were increased in decidual cells compared with endometrial cells. Endometrial cells were used as the negative control and endometrial cells treated with

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cAMP +P4 served as the positive control (Enzo Life Sciences, NY, USA) (Gellersen & Brosens, 2003) (Figure 1).

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2.2. Endometrial epithelial cell culture

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Suspensions of endometrial cells were obtained by enzymatic digestion and mechanical means, as described previously (Chan et al., 2004; Seli et al, 2001). The endometrium

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was scraped off from the underlying myometrium, finely diced, and dissociated in

streptomycin (200 μg/ml), and collagenase (1 mg/ml [15 U mg/mg]; Sigma) for 30 min

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Hanks7 balanced salt solution containing HEPES (25 mM), penicillin (200 U/ml),

at 37°C with agitation (Kato et al., 2007). The dispersed endometrial cells were

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separated from debris by filtration through a wire sieve (32 μm diameter pores).

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Endometrial cells were cultured as a cell line. This was used in the non-pregnant group

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2.3. Total RNA extraction

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(Kato et al., 2007).

Total RNA from decidual cells was extracted using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions for QIAshredder (50)

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(Qiagen, Valencia, CA, USA). The total RNA concentration was determined by 165

ultraviolet absorbance at 260 nm (A260) via direct measurement on a NanoDrop ND2000 spectrophotometer (ThermoFisher Scientific, Wilmington, DE, USA). The purity of RNA was assessed by evaluating readings at 260 and 280 nm (A260/A280), which provides an estimate of the purity of RNA with respect to contaminants that absorb UV, such as 9 Page 9 of 40

protein. All samples had A260/280 values > 2.0, thus indicating a high level of purity.

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2.4. Microarray analysis of decidual primary culture

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Microarray analysis was performed by Takara Bio (Tokyo, Japan) customer services.

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The integrity of the RNA was checked using an RNA 6000 Nano Assay kit and 2100

100 ng of cDNA of decidual cells was hybridized for 17 h at 65°C on Gene Chip SurePrint G3 Human GE (8×60K,

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Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Following fragmentation,

G4851A; Agilent Technologies); washing was

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performed in accordance with the instructions of the manufacturer.

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For DNA microarray analyses, One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling, version 6.0) using SurePrint G3 Human GE and an in 180

situ Hybridization Kit (Agilent Technologies) was used according to the manufacturer’s protocols.

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The fluorescence-labeled cRNA samples were fragmented randomly, hybridized to SurePrint G3 Human GE, and then incubated at 65°C for 17 h in a rotisserie oven set at 10 rpm. Following hybridization, the microarrays were washed using Agilent wash 185

buffers according to the wash protocol, and were scanned using an Agilent Scanner.

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Array quality was assessed through the use of control features, as well as spike-in controls (One-Color Spike-in Kit; Agilent Technologies).

The resulting text files

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extracted from the Feature Extraction software were imported into GeneSpring GX

intensity was cut off before normalization. The microarray data sets were normalized in

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11.5.1 software (version 10.7.1.1; Agilent Technologies) for further analysis. Background

GeneSpring GX using the Agilent FE one-color scenario (primarily 75% normalization),

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and the normalized signals were logarithmically (base 2) transformed for hierarchical

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clustering analysis of strain differences of the vehicle control group. The Detected-Compromised-Not detected flag was used to identify and omit genes with

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probes of poor quality or low expression values.

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2.5. Pathway analysis

Genetic pathways were evaluated using the Meta Core analysis Suite (Gene Go).

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Enrichment analysis consisted of mapping gene identities (IDs) of the dataset onto IDs 200

in entities of built-in functional ontologies represented in Meta Core by pathway maps and networks.

2.6. Quantitative real-time RT-PCR measurement 11 Page 11 of 40

Quantitative real-time RT-PCR was performed employing the 7500 Fast Real-Time PCR 205

System. Expression levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8 mRNA were

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measured using quantitative real-time RT-PCR in the decidual cells, cultured in

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serum-free media for 24 h before treatment with P4, IL-1β, and IL-1β + P4. SYBR

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premix Ex Taq (TAKARA, Tokyo, Japan) was used for cDNA quantification.

Standard curves for both the genes of interest and GAPDH were generated with serial dilutions of pooled cDNA samples. The amplification efficiency for each primer set was

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determined by converting the slope of the standard curve using algorithm E. For each

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gene, the mean threshold cycle (from duplicate reactions) was corrected for the efficiency of the reaction and expressed relative to a control sample for each experiment.

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Each gene of interest was then expressed relative to GAPDH levels. The list of primers used for PCR is as follows.

The primers for GAPDH were 5’-GAAGGTGAAGGTCGGAGTC-3’ (sense) and

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5’-GAAGATGGTGATGGGATTTC-3’ (antisense).

The primers for PTGS-2 were 5’-TACTTGGCCACTATCGACTGG-3’ (sense) and 5’-GCCGTGTGACTTACAGATGGT-3’ (antisense).

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The primers for PTGFR were 5’-TCCTGTATTTGTTGGAGCCCATTTCTGGTT-3’

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(sense) and 5’-TCCATGTTGCCATTCGGAGAGCAAAAAG-3’ (antisense).

The primers for NFκ-B p65 were 5’-CTGCCCGGGATGGCTTCTAT-3’ (sense) and

for

IL-17

were

5’-GTCTGGGCGCAGGTATGTGG-3’

(sense)

and

5’-CACCGTGGAGACCCTGGAGGC-3’ (antisense).

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primers

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The

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5’-CCGCTTCACACACTGGAT-3’ (antisense).

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The primers for IL-8 were 5’-ATGACTTCCAAGCTGGCCGTGGCT-3’ (sense) and

2.7. Western blot analysis

Confluent decidual cells were cultured in serum-free medium for 24 h before treatment

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5’-TCTCAGCCCTCTTCAAAAACTTCTC-3’ (antisense).

with IL-1β, P4, and IL-1β + P4.

Decidual cells were homogenized on ice in 300 μl of lysis buffer (Sigma-Aldrich)

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containing Protease Inhibitor Cocktail (Sigma-Aldrich).

After centrifugation at 20,000 g × for 10 min at 4°C, the protein concentration of the 235

supernatant was determined using a bicinchoninic acid (BCA) Protein Assay Kit (Thermo Scientific, Pierce Biotechnology, Rockford, IL, USA). Ten micrograms of each

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protein sample was used for Western blot analysis. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating

The membranes were blocked with 5% skim milk in 0.1% TBS Tween for 1 h. To detect

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and 4% stacking gels, and transferred to nitrocellulose membranes.

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phospho-IκBα, the membranes were blocked with 1% bovine serum albumin in TBS with 0.05% Tween-20 for 1 h, followed by incubation overnight at 4°C with specific

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antibodies to Iκ-Bα (Cell Signaling Technology, Beverly, MA, USA), NFκ-B p65 (Cell

p21(187) (Santa Cruz Biotechnology), and β-actin (Santa Cruz Biotechnology). The same

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Signaling Technology), Phospho-IκBα (Ser32/36) (5A5) (Cell Signaling Technology),

blots were used for the quantitative analyses of each target protein. β-actin served as a

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loading control. The membranes were washed and then incubated in appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactivity was detected using the ECL detection kit (GE Healthcare, Little Chalfont, 250

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Buckinghamshire, UK) according to the manufacturer’s instructions. Semi-quantitative densitometric analysis of the immunoreactive bands (intensity X area) was performed using MultiGauge software (FUJI FILM, Tokyo, Japan).

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2.8. Senescence-associated-β-galactosidase staining

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Senescence was determined histochemically in treated and untreated control cells

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employing a Senescence Detection Kit (Bio Vision) according to the manufacturer’s

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instructions. We counted the percentage of senescence-associated-β-galactosidase

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(SA-β-gal)-positive cells, identified by intense blue staining, as reported previously

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(Brown et al., 1997; Dimri et al., 1995; Hirota et al., 2010; Krizhanovsky et al., 2008).

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2.9. Statistical analysis

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The relationships between two groups of findings and numerical values obtained by

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real-time PCR and other assays were analyzed using the Mann–Whitney U-test. The non-adjusted statistical level of significance for all p values was <0.05.

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3. Results

3.1. Gene expressions in decidual cells treated with IL-1β or IL-1β + P4 We have previously shown that PTGFR and PTGS-2 mRNA (Makino et al., 2009) changed with additional IL-1β and P4; thus, we performed microarray analysis on 270

decidual cells (passages 3–6) to examine the genes that are regulated by IL-1β or P4 15 Page 15 of 40

using a sample in which PTGFR and PTGS-2 mRNA expression showed significant changes (Table 2a–d).

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The top 30 genes up-regulated by IL-1β and IL-1β + P4, based on Gene Symbol, Gene

b). The top 30 genes down-regulated by IL-1β and IL-1β + P4 are shown in Table 1c, d.

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Name, Genbank Accession, and Fold Change in decidual cells, are presented (Table 2a,

Frequently up-regulated (relative to normal decidual cells) genes in decidual cells

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treated with IL-1β were CSF3, CXCL6, and IL-8, all of which are inflammatory genes

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(Table 2a). The top 30 genes up-regulated in response to treatment with IL-1β + P4 in

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decidual cells were different from those that were up-regulated by IL-1β alone (Table 2b). The expression of IL-8, an inflammatory gene, was down-regulated in response to

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IL-1β + P4 (Table 2d), and up-regulated following exposure to IL-1β alone (Table 2a). Genetic pathways were evaluated using the Meta Core Analysis Suite (Gene Go.) treated with IL-1β alone (Figure 2a, b) and IL-1β + P4 (data not shown). IL-17 and

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NFκ-B signaling pathways are highly expressed in decidual cells treated with IL-1β 285

alone (Figure 2a, b).

In contrast, NFκ-B was not up-regulated and IL-8 was

down-regulated by IL-1β + P4 (data not shown).

3.2. Evaluation of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8 mRNA expression in decidual cells 16 Page 16 of 40

Real-time PCR revealed low expression levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, 290

and IL-8 mRNA in untreated human decidual cells. In decidual cells treated with IL-1β,

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the levels of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8 mRNA were significantly

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increased compared with the cells treated with the vehicle (p<0.05; Figure 3), and

3.3 Levels of Iκ-Bα, NFκ-B p65, and phospho-IκBα protein in decidual cells

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IL-1β + P4 treatment (Figure 3a–e) .

Iκκ activation by phosphorylation targets Iκ-Bα for ubiquitination and degradation by

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the proteasome either via the canonical pathway or by p100 processing to p52 in the

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noncanonical pathway. Subsequently, phosphorylated NFκ-B dimers bind to κB DNA elements and induce transcription of target genes. Therefore, we analyzed the levels of 300

Iκ-Bα, NFκ-B p65, and phospho-IκBα using Western blot analysis to assess NFκ-B pathway activity (Figure 4a). The Iκ-Bα/β-actin ratio (Figure 4b) and NFκ-B p65/β-actin

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ratio (Figure 4c) were not significantly changed; however, the phospho-IκBα/Iκ-B ratio was increased by IL-1β, and it was significantly decreased by IL-1β + P4 (p<0.05) to a similar level in untreated decidual cells (Figure 4d).

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3.4 SA-β-gal positivity and senescence were induced by treatment of decidual cells with IL-1β

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We next investigated whether IL-1β induced senescence in decidual cells. The

IL-1β than those exposed only to the vehicle (p < 0.05; Figure 5a). In contrast, the

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proportion of SA-β-gal-positive cells was significantly higher in the cells treated with

number of SA-β-gal-positive cells was not affected by IL-1β in endometrial cell cultures

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(Figure 5b). The level of p21 was increased in cells treated with IL-1β compared with

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cells treated with vehicle and P4 in decidual cells (Figure 5c). Thus, senescence was

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induced in decidual cells by the addition of IL-1β, but not in normal endometrial cells.

4. Discussion

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Risk factors for preterm delivery include maternal characteristics, prior obstetrical history of preterm delivery, medical complications, genetic predisposition, and

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environmental factors. Prostaglandins are also pivotal to the parturition process, 320

mediating cervical ripening and dilatation and stimulating myometrial contractions (Lei et al., 2011; Olson, 2003). The generation of prostaglandins by amnion and decidual tissues is critical for both the initiation and the continuance of human labor.

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Bowen et al. (2002) determined cytokine levels in tissue extracts from preterm deliveries and concluded that IL-1β, IL-6, and IL-8 concentrations in decidua are elevated in spontaneous preterm deliveries.

Menon et al. (1995) determined the

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expression of inflammatory cytokines (IL-1β and IL-6) by fetal membranes in response

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to infection in vivo and to endotoxin in organ cultures. Increased secretion of IL-8 from placenta with chorioamnionitis has also been reported (Shimoya et al., 1992).

women not in labor that was higher than in deciduas of women who underwent labor at

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We observed a late gestation increase in PTGFR mRNA and protein in the deciduas of

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term (Makino et al., 2007). Thus, considering the interaction between P4 and PTGFR expression, P4 may play an important role in suppressing the expression of PTGFR,

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thereby maintaining uterine quiescence. We have also confirmed that knockdown of PTGFR by siRNA had no effect on the expression of PTGS-2 mRNA. Thus, PTGFR was 335

thought to not be present up-stream from PTGS-2 (data not shown).

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We have previously shown that changes in PTGFR and PHGS-2 mRNA had a similar pattern to the protein levels in decidua cells (Makino et al., 2007); thus, the present study focused on mRNA as a preliminary study.

Genes up-regulated by IL-1β treatment were associated with modulators of the immune 340

response, including the IL-17 signaling and NFκ-B signaling pathways. Genes 19 Page 19 of 40

up-regulated by IL-1β + P4 treatment are not related to inflammatory pathways (Hillier et al.,1995). The differences in up-regulated genes between IL-1β + P4 and IL-1β alone

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suggest that P4 also changes the suppression of genes, thereby creating an environment

NFκ-B is a transcription factor regulating the gene expression of growth factors,

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favoring anti-inflammatory processes.

numerous cytokines, and enzymes involved in a variety of pivotal cellular processes,

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including responses to inflammation and oxidative stress (Hayden & Ghosh, 2008). In

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mammals, the NFκ-B family is composed of five members including p50/NFκ-B1,

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NFκ-B2, p65/RelA, RelB, and c-Rel. NFκ-B is retained in an inactive form in the cytoplasm by binding to a member of the IκB family (Huang et al.; Oh et al., 2004). The

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actions of IL-1β are mediated by several transcription factors, including members of the NFκ-B family for amnion epithelial cells, and are derived simultaneously with mesenchymal cells (Okita et al., 1983; Ackerman et al., 2008). The contribution of

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NFκ-B to human labor has been addressed (Lindström & Bennett, 2005). While 355

biochemical and immunohistochemical results have been conflicting, the present consensus suggests that NFκ-B activation might both precede and accompany parturition. However, there have been no genomic or associative genetic reports directly linking the NFκ-B pathway to labor. Our findings support the role of the NFκ-B 20 Page 20 of 40

pathway in the induction of labor.

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The p21 is known to be involved in terminal cell differentiation and senescence in other

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systems (Chen et al., 2005). Senescence-associated growth restriction with increased

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levels of p21 in decidual cells, possibly by IL-1β, may participate in the processes of

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preterm labor independently (Brown et al., 1997; Dimri et al., 1995; Hirota et al., 2010; Krizhanovsky et al., 2008). Our results suggest that P4 may block the effects of IL-1β on senescence.

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As a limitation of this paper, the present study examined mRNA expression and has not

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addressed changes in proteins. However, we have already reported changes in PTGFR

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and PHGS-2 mRNA in decidual cells (Makino et al., 2009).

Although the molecular mechanisms responsible for human parturition remain 370

incompletely understood, there is abundant evidence to suggest that inflammation might play an important role in this process. P4 has been shown to be essential for the

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maintenance of pregnancy in humans. In the future, selective inhibitors for IL-1β, NFκ-B p65, and p21 may represent new pharmacological interventions for inflammation.

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In summary, our studies have demonstrated that P4 changes the gene expression levels

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in a manner that favors an anti-inflammatory milieu. As IL-8 appeared to be the cytokine whose expression is most significantly modulated by P4, further studies

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evaluating IL-8 as a therapeutic target are needed.

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Acknowledgements

We thank Dr. Tsutomu Fujimura and Dr. Reiko Mineki, Ms. Tomomi Ikeda, Ms. Takako

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Ikegami, and Ms. Akemi Matsumoto for their excellent technical assistance. And we

assistance.

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thank Dr. Keiji Kuroda, Dr. Yoshihiko Araki and Prof. David M Olson for writing

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Funding source

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JAOG-Ogyaa-donation foundation

Conflict of interest

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The authors have no conflict of interest.

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Huang, S. J., Schatz, F., Masch, R., Rahman, M., Buchwalder, L., Niven-Fairchild, T., et al. 2006. Regulation of chemokine production in response to pro-inflammatory cytokines in first trimester decidual cells. J Reprod Immunol, 72 , 60-73.

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Interleukin-1 beta-stimulated PGE2 production from early first trimester human decidual cells is inhibited by dexamethasone and progesterone. Prostaglandins, 49 , 15-26. Kato, K., Yoshimoto, M., Adachi, S., Yamayoshi, A., Arima, T., Asanoma, K., et al. 2007. Characterization of side-population cells in human normal endometrium. Hum 25 Page 25 of 40

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actions of progestins to inhibit cervical ripening and prevent delivery depend on their properties, the route of administration, and the vehicle. Am J Obstet

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labour. Reproduction, 130 , 569-581.

Lucovnik M, Kuon RJ, Chambliss LR, Maner W, Shi SQ, Shi L et al., 2011. Progestin 485

treatment for the prevention of preterm birth. Acta Obstet Gynecol Scand, 90,

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decidua at preterm birth and term birth. Placenta, 28 , 557-565. Makino S, Matsuoka K, Aidiresi A, Seki H, Takeda S. 2009. Regulation of prostaglandin

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F2a receptor (FP) mRNA in human decidua cells during pregnancy and parturition. Supplement of Reproductive Science. 16 , 294.

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Obstet Gynecol, 47 , 784-795; discussion 881-782. Meis, P. J., Klebanoff, M., Thom, E., Dombrowski, M. P., Sibai, B., Moawad, A. H., et al., 2003. Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med, 348 , 2379-2385. Meis, P. J., Klebanoff, M., Dombrowski, M. P., Sibai, B. M., Leindecker, S., Moawad, A.

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human p53 gene through induction of NF-kappaB activation cascade. Oncogene, 23 , 8282-8291.

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Romero, R., Yeo, L., Miranda, J., Hassan, S. S., Conde-Agudelo, A., & Chaiworapongsa, T. 2013. A blueprint for the prevention of preterm birth: vaginal progesterone in

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Seli, E., Senturk, L. M., Bahtiyar, O. M., Kayisli, U. A., & Arici, A. 2001. Expression of aminopeptidase N in human endometrium and regulation of its activity by estrogen. Fertil Steril, 75 , 1172-1176. Shimoya, K., Matsuzaki, N., Taniguchi, T., Jo, T., Saji, F., Kitajima, H., et al., 1992. 535

Interleukin-8 in cord sera: a sensitive and specific marker for the detection of preterm chorioamnionitis. J Infect Dis, 165 , 957-960. 27 Page 27 of 40

Tromp, G., Kuivaniemi, H., Romero, R., Chaiworapongsa, T., Kim, Y. M., Kim, M. R., et al., 2004. Genome-wide expression profiling of fetal membranes reveals a deficient expression of proteinase inhibitor 3 in premature rupture of 540

membranes. Am J Obstet Gynecol, 191, 1331-1338. Willman, E. A., & Collins, W. P. 1976. Distribution of prostaglandins E2 and F2 alpha

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Figure Legends Fig. 1: The levels of prolactin receptor (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1) were measured using Western blot analysis. 550

The levels of PRL and IGFBP-1 were increased in decidual cells compared with

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endometrial cells. Fig. 2: Pathway analysis.

Microarray expression analysis for screening up-regulated genes induced by IL-1β (a). We used the Metacore package to identify the Gene Go Pathways Maps involved in

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IL-17 signaling pathway was mostly responsible (b).

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up-regulated genes in the microarray data on a set of treatment with IL-1β (b). The Experimental data from all files are linked to and are visualized on the maps as thermometer-like figures. Upward thermometers are red and indicate up-regulated signals, and downward blue thermometers indicate down-regulated levels of expression

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Fig. 3: Expression of PTGS-2, PTGFR, NFκ-B p65, IL-17, and IL-8 mRNA in decidual cells by RT-PCR analysis.

Levels of expression of (a) PTGS-2, (b) PTGFR, (c) NFκ-B p65, (d) IL-17, and (e) IL-8

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mRNA in human decidual cells under various conditions, in the presence or absence of P4 (10-6 M) and IL-1β (5 ng/ml; 100 μM/ml) are shown by columns. The difference in IL-8

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expression was most pronounced when treatment with IL-1β alone was compared with IL-1β + P4 treatment. The housekeeping gene GAPDH was used as an internal control. 570

The results are representative of eight independent experiments; the vertical bar indicates SD. Statistical significance between the two groups is indicated by an asterisk.

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Fig. 4: Iκ-Bα, NFκ-B p65, and phospho-IκBα proteins in cultured decidual cells by 575

Western blot analysis. The levels of Iκ-Bα, NFκ-B p65, and phospho-IκBα by Western blot analysis in the presence/absence of IL-1β (5 ng/ml) and P4 (10-6 M) (a). Relative levels of expression of Iκ-Bα (b) and NFκ-B p65 (c) normalized to the level of β-actin expression. The level of phospho-IκBα expression was indicated as the relative intensity compared with that of

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Iκ-Bα (d). Data are the mean with SD from four independent experiments. Statistical significance between two groups is indicated by an asterisk.

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Fig. 5: Induction of senescence in cultured cells monitored with senescence associated-beta-galactosidase (SA-β-gal) staining. 585

The results are representative of three independent experiments. Fig. 5a) IL-1β (5 ng/ml) treatment resulted in the appearance of SA-β-gal-positive cells, as demonstrated by cytoplasmic blue staining. The proportion of SA-β-gal-positive cells

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increased when treated with IL-1β compared with treatment with vehicle, P4 (10-6 M), and IL-1β + P4 (p<0.05). We counted 200 cells in 10 microscopic fields to determine the 590

percentage of SA-β-gal-positive cells. Scale bar: 100 μm.

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Fig. 5b) The level of SA-β-gal positivity and senescence was not significant by treatment of normal endometrial cells with IL-1β, P4, and IL-1β + P4. Scale bar: 100 μm.

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Fig. 5c) We performed Western blot analysis to investigate p21 protein in decidual cells. The level of p21 protein was increased in decidual cells treated with IL-1β compared

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with cells treated with vehicle, P4, and IL-1β + P4 (p<0.05).

Patient characteristics, following the inclusion criteria

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Total number Median age in years (range) Weeks of gestation (weeks)

34.5 (20-40) 37-38

Exclusion criteria include:

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FGR, placenta previa, PROM, fetal anomaly, and bleeding

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GeneName

GenbankAccession Fold Change

Table 2a

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colony stimulating factor 3 (granulocyte) NM_000759 chemokine (C-X-C motif) ligand 6 (granulocyte chemotactic protein 2) NM_002993 colony stimulating factor 3 (granulocyte) NM_000759 interleukin 8 NM_000584 chemokine (C-X-C motif) ligand 3 NM_002090 complement component 3 NM_000064 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)NM_001511 interleukin 1, beta NM_000576 chemokine (C-C motif) ligand 5 NM_002985 chemokine (C-X-C motif) ligand 2 NM_002089 chemokine (C-X-C motif) ligand 2 NM_002089 chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)NM_001511 tau tubulin kinase 1 NM_032538 leucine rich repeat containing 15 NM_130830 prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) NM_000963 chemokine (C-C motif) ligand 7 NM_006273 G protein-coupled receptor 37 like 1 NM_004767 chemokine (C-C motif) ligand 8 NM_005623 hydroxysteroid (11-beta) dehydrogenase 1 NM_181755 colony stimulating factor 2 (granulocyte-macrophage) NM_000758 adenosine A2a receptor NM_000675 serpin peptidase inhibitor, clade B (ovalbumin), member 3 NM_006919 2'-5'-oligoadenylate synthetase 1, 40/46kDa NM_002534 chondroitin sulfate N-acetylgalactosaminyltransferase 1 NM_001130518 interleukin 6 (interferon, beta 2) NM_000600 ubiquitin D NM_006398 chromosome 15 open reading frame 48 NM_032413 interleukin 1 receptor antagonist NM_173843 guanine nucleotide binding protein (G protein), alpha 15 (Gq class) NM_002068 chemokine (C-C motif) ligand 20 NM_004591

12.803 11.561 11.391 11.023 10.291 10.037 10.015 9.891 9.843 9.642 9.536 9.322 9.074 9.013 8.355 8.149 8.070 8.020 7.908 7.816 7.782 7.744 7.741 7.608 7.526 7.484 7.456 7.371 7.330 7.267

Top 30 up-regulated genes by IL-1β with gene symbol, gene name, Genbank accession number, and

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1 2 3 Gene Symbol 4 CSF3 5 CXCL6 6 CSF3 7 IL8 8 CXCL3 9 10 C3 11 CXCL1 12 IL1B 13 CCL5 14 CXCL2 15 CXCL2 16 CXCL1 17 TTBK1 18 19 LRRC15 20 PTGS2 21 CCL7 22 GPR37L1 23 CCL8 24 HSD11B1 25 CSF2 26 ADORA2A 27 28 SERPINB3 29 OAS1 30 CSGALNACT1 31 IL6 32 UBD 33 C15orf48 34 IL1RN 35 GNA15 36 37 CCL20 600 38 39 40 41 42 43 44 45 605 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

fold change in decidual cells.

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1 2 Gene Symbol GeneName GenbankAccession Fold Change 3 4 LOC646471 uncharacterized LOC646471 NR_024498 4.555 5 CES1P2 carboxylesterase 1 pseudogene 2 NR_033740 4.415 6 C9orf66 chromosome 9 open reading frame 66 NM_152569 4.318 7 DCDC5 doublecortin domain containing 5 NM_020869 4.242 8 C3orf49 chromosome 3 open reading frame 49 NR_026866 4.209 9 LINC00272 long intergenic non-protein coding RNA 272 NR_034131 4.120 10 11 NRK Nik related kinase BC108702 4.077 12 C4orf51 chromosome 4 open reading frame 51 NM_001080531 4.032 13 OR4D9 olfactory receptor, family 4, subfamily D, member 9 NM_001004711 3.988 14 C8orf74 chromosome 8 open reading frame 74 NM_001040032 3.959 15 DCAF8L1 DDB1 and CUL4 associated factor 8-like 1 NM_001017930 3.810 16 MYADML2 myeloid-associated differentiation marker-like 2 NM_001145113 3.776 17 CRYBB3 crystallin, beta B3 NM_004076 3.774 18 19 DPY19L2 dpy-19-like 2 (C. elegans) NM_173812 3.721 20 IZUMO1 izumo sperm-egg fusion 1 NM_182575 3.675 21 CLEC4GP1 C-type lectin domain family 4, member G pseudogene 1 NR_002931 3.646 22 ZNF717 zinc finger protein 717 NM_001128223 3.622 23 LOC286114 uncharacterized LOC286114 NR_033894 3.561 24 LOC255167 uncharacterized LOC255167 NR_024424 3.471 25 LOC151300 uncharacterized LOC151300 NR_024385 3.389 26 27 NEUROG1 neurogenin 1 NM_006161 3.373 28 LOC100128511uncharacterized LOC100128511 XR_109015 3.337 29 CAMSAP3 calmodulin regulated spectrin-associated protein family, member 3 NM_001080429 3.305 30 RAB39B RAB39B, member RAS oncogene family NM_171998 3.295 31 OAS1 2'-5'-oligoadenylate synthetase 1, 40/46kDa NM_002534 3.288 32 COBL cordon-bleu homolog (mouse) NM_015198 3.283 33 34 POLR2F polymerase (RNA) II (DNA directed) polypeptide F XR_109712 3.271 35 CTAGE1 cutaneous T-cell lymphoma-associated antigen 1 NM_172241 3.192 36 SLC35F1 solute carrier family 35, member F1 NM_001029858 3.160 37 OR52B6 olfactory receptor, family 52, subfamily B, member 6 NM_001005162 3.151 38 39 40 Table 2b 41 42 Top 30 up-regulated genes by IL-1β + P4 with gene symbol, gene name, Genbank accession number, 43 610 and fold change in decidual cells. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 32 62 63 64 Page 32 of 40 65

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1 2 Gene Symbol GeneName GenbankAccession Fold Change 3 4 ANGPT2 angiopoietin 2 NM_001147 -4.427 5 HSD17B2 hydroxysteroid (17-beta) dehydrogenase 2 NM_002153 -4.232 6 HTR3D 5-hydroxytryptamine (serotonin) receptor 3 family member D NM_001145143 -4.160 7 H2BFWT H2B histone family, member W, testis-specific NM_001002916 -4.079 8 PRELP proline/arginine-rich end leucine-rich repeat protein NM_002725 -4.000 9 ANKRD18B ankyrin repeat domain 18B NM_001244752 -3.952 10 11 ZAR1 zygote arrest 1 NM_175619 -3.950 12 FLJ43390 uncharacterized LOC646113 NR_015358 -3.890 13 DDIT4L DNA-damage-inducible transcript 4-like NM_145244 -3.814 14 XLOC_003491 -3.811 15 SYTL5 synaptotagmin-like 5 NM_138780 -3.780 16 LOC100128054uncharacterized LOC100128054 NR_033969 -3.722 17 DDIT4L DNA-damage-inducible transcript 4-like BC013592 -3.677 18 19 ANXA3 annexin A3 NM_005139 -3.651 20 LOC100131821uncharacterized LOC100131821 AK123052 -3.650 21 KCNC1 potassium voltage-gated channel, Shaw-related subfamily, member 1 NM_004976 -3.647 22 COPG2IT1 COPG2 imprinted transcript 1 (non-protein coding) NR_024086 -3.645 23 SPTB spectrin, beta, erythrocytic NM_001024858 -3.622 24 ATP6V0D2 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d2 NM_152565 -3.605 25 TTC39A tetratricopeptide repeat domain 39A NM_001080494 -3.590 26 27 ROR1 receptor tyrosine kinase-like orphan receptor 1 AK000776 -3.563 28 STMN2 stathmin-like 2 NM_007029 -3.552 29 PADI1 peptidyl arginine deiminase, type I NM_013358 -3.520 30 KRTAP2-2 keratin associated protein 2-2 NM_033032 -3.509 31 SLC14A1 solute carrier family 14 (urea transporter), member 1 (Kidd blood group) NM_001146037 -3.500 32 OR2A5 olfactory receptor, family 2, subfamily A, member 5 NM_012365 -3.494 33 34 ITIH3 inter-alpha-trypsin inhibitor heavy chain 3 NM_002217 -3.465 35 OSR1 odd-skipped related 1 (Drosophila) NM_145260 -3.443 36 C8orf45 chromosome 8 open reading frame 45 NM_001136160 -3.424 37 OR7E37P olfactory receptor, family 7, subfamily E, member 37 pseudogene NR_002163 -3.412 38 39 40 Table 2c 41 42 Top 30 down-regulated genes by IL-1β with gene symbol, gene name, Genbank accession number, 43 615 and fold change in decidual cells. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 33 62 63 64 Page 33 of 40 65

GenbankAccession

5-hydroxytryptamine (serotonin) receptor 3 family member D zygote arrest 1 ankyrin repeat domain 18B centromere protein N uncharacterized LOC100131821 spectrin, beta, erythrocytic metallophosphoesterase domain containing 1 chromosome 8 open reading frame 45 H2B histone family, member W, testis-specific DNA-damage inducible 1 homolog 1 (S. cerevisiae) olfactory receptor, family 7, subfamily E, member 37 pseudogene disabled homolog 1 (Drosophila) crumbs homolog 2 (Drosophila) Smith-Magenis syndrome chromosome region, candidate 5 (non-protein coding) ring finger protein 144B pancreatic lipase-related protein 3 uncharacterized LOC100132707 chromosome 9 open reading frame 41 olfactory receptor, family 1, subfamily S, member 2 period homolog 3 (Drosophila) pseudogene chemokine (C-C motif) ligand 3 uncharacterized LOC100132147 RasGEF domain family, member 1B ankyrin 3, node of Ranvier (ankyrin G) transcription factor NF-E4 interleukin 8 WW and C2 domain containing 2 unc-93 homolog A (C. elegans) hect domain and RLD 2 pseudogene 7 chordin-like 2

NM_001145143 NM_175619 NM_001244752 NM_001100625 AK123052 NM_001024858 NM_001044370 NM_001136160 NM_001002916 NM_001001711 NR_002163

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NM_173689 NR_024007 NM_182757 NM_001011709 BC050402

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Table 2d

Fold Change

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GeneName

NM_001004459 NR_002790 NM_002983 BC036435 NM_152545 NM_001204404 NM_001085386 NM_000584 AK126057 NM_018974 AF071178 NM_015424

-4.038 -3.838 -3.830 -3.692 -3.530 -3.511 -3.458 -3.305 -3.297 -3.290 -3.215 -3.105 -2.985 -2.846 -2.837 -2.824 -2.787 -2.782 -2.779 -2.747 -2.740 -2.710 -2.708 -2.681 -2.675 -2.674 -2.673 -2.651 -2.630 -2.547

Top 30 down-regulated genes by IL-1β + P4 with gene symbol, gene name, Genbank accession number, and fold change in decidual cells.

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1 2 Gene Symbol 3 4 HTR3D 5 ZAR1 6 ANKRD18B 7 CENPN 8 LOC100131821 9 SPTB 10 MPPED1 11 C8orf45 12 H2BFWT 13 DDI1 14 15 OR7E37P 16 DAB1 17 CRB2 18 SMCR5 19 RNF144B 20 PNLIPRP3 21 LOC100132707 22 C9orf41 23 OR1S2 24 PER4 25 CCL3 26 LOC100132147 27 28 RASGEF1B 29 ANK3 30 NFE4 31 IL8 32 WWC2 33 UNC93A 34 HERC2P7 35 CHRDL2 36 37 38 39 40 620 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Figure1

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Figure2

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Figure3

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Figure4

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Figure5

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*Conflict of Interest Statement

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Dummy conflict of interest statement to allow submission of copyedited version.

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