Exposure to cigarette smoke affects endometrial maturation including angiogenesis and decidualization

Abstract Purpose To elucidate the effects of cigarette smoking on human endometrial maturation for reproductive function, the authors examined the in vitro effects of cigarette smoke extract (CSE) on angiogenesis and decidualization in primary human endometrial stromal cells (ESCs). Methods Endometrial stromal cells were cultured with CSE and/or estradiol‐17β (E2) and medroxyprogesterone acetate (MPA). The mRNA, protein levels, and protein secretion of the angiogenic factors and decidual specific factors were assessed using real‐time polymerase chain reaction, Western blot analysis, and enzyme‐linked immunosorbent assay, respectively. Decidualization was also monitored by the changes in cellular morphology. Results Endometrial stromal cell proliferation substantially decreased after dose‐dependent treatments with CSE at concentrations above 1%, whereas cell death was induced at treatment concentrations above 1% CSE. Treatments above 0.025% CSE led to increased vascular endothelial growth factor mRNA through hypoxia‐inducible factor‐1α accumulation. CSE concentrations at 0.01% and 0.025% increased the prolactin expression levels after treatment with E2 and MPA, whereas 0.1% and 0.25% CSE concentrations suppressed prolactin. Similar tendencies were observed in cellular morphology and other decidual specific factors. Conclusion These results suggest that exposure to cigarette smoke affects endometrial appropriate maturation including the processes of angiogenesis and decidualization in the reproductive system.


| INTRODUC TI ON
Smoking and passive smoking affects the establishment and maintenance of pregnancy. The association between tobacco consumption and female infertility in natural cycles is consistently reported in epidemiological studies. 1 The relationships between ovarian luteal (endocrine) functional factors, endometrial (maternal) factors, and embryo (fetal) factors are important for establishing pregnancy. The effect of smoking on ovarian endocrine function is associated with a lower average age of menopausal women compared to non-smokers. 2 Studies on in vitro fertilization cycles have demonstrated that cigarette smoking appears to significantly reduce their ovarian reserve and result in a poor response to ovarian stimulation at an earlier age. 3 On the other hand, the effect of smoking on the fetal factor is reported to show deleterious changes in the placenta and fetus and is more frequent in pregnant smokers who present higher rates of low birthweight and perinatal and neonatal mortality. 4 However, as far as we know, the effects of smoking on the human endometrium remain poorly understood.
Angiogenesis also plays a central role in endometrial function, as improper vascularization of the endometrium may cause implantation failure and infertility. 5,6 Several angiogenic factors in the human endometrium have been identified as important regulators of physiological angiogenesis 7,8 including vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1, also known as CXCL12), and angiopoietin 1 and 2 (ANGPT1 and 2). [9][10][11] VEGF is a key mediator of physiological and pathological vascular remodeling. 12 SDF-1 functions as a potent inducer of angiogenesis, stimulating endothelial cell proliferation and cell survival. 13 ANGPT1 and 2 are a second key group of promotors of angiogenesis and vessel remodeling on the endometrium and interact with VEGF. 14 The human endometrium undergoes periodic proliferation and differentiation that is controlled during the menstrual cycle by a continuous and careful interaction of the ovarian steroids including estradiol-17β (E 2 ) and progesterone. 15 Decidualization is an essential process in the differentiation of endometrial stromal cells (ESCs) that is accompanied by dramatic changes in cell function. The decidual reaction plays a central role in embryo implantation and pregnancy establishment. 16 Many studies have investigated the regulation and the molecular mechanisms involved in decidualization using human-cultured ESCs in vitro. Morphologically, decidualization is characterized by the transformation of elongated fibroblast-like ESCs into enlarged round cells with specific structural modifications.
The exposure of ESCs to progestin for 12 days, alone or in combination with estrogen, triggers the expression of decidual specific factors such as prolactin (PRL) and insulin-like growth factor-binding protein 1(IGFBP-1). Previously, we demonstrated that progestin increased heart and neural crest derivatives-expressed transcript 2 (HAND2) expression during ESC decidualization. 17 HAND2 is a transcription factor required for the growth and development of the heart, branchial arches, and limb buds. Recent reports have demonstrated that progestin-induced HAND2 regulates decidual specific genes including interleukin-15 (IL-15) and fibroblast growth factor 9 (FGF9). 18,19 In this study, we aimed to clarify the direct effects of smoking on endometrial angiogenesis and decidualization using a human ESCs and a cigarette smoke extract (CSE) an in vitro model.

| Tissue collection
Human tissues were obtained after written informed consent from each patient in accordance with the Declaration of Helsinki. Human endometrial tissues in the proliferative phase were obtained from 27 women aged 32-47 years, who underwent hysterectomies due to uterine fibroids. The patients had regular menstrual cycles and no preoperative hormonal treatment. A section of each endometrial specimen was analyzed and confirmed to be histologically normal. This study was approved by the institutional review board of Kansai Medical University, Osaka, Japan (project approval number 2006101).

| Cell culture and treatment
ESCs were purified from the endometrial tissues using a standard enzyme digestion method described previously. 20 ESCs were cultured in DMEM/F-12 supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL penicillin, 100 mg/mL streptomycin, and 0.25 μg/mL Amphotericin B (Antibiotic-Antimycotic 100×; GIbco, Grand Island, NY, USA, MA, USA) at 37°C in a humidified atmosphere of 5% CO 2 . The culture medium was replaced 60 minute after plating to minimize epithelial cell contamination. The percentage of vimentin-positive cells in the confluent ESCs was confirmed to be >99% according to immunohistochemical staining, as described previously. 21 After passage 0-1, when ESCs were nearly confluent, the cells were trypsinized and re-plated. To negate the effect of endogenous steroid hormones, cells were cultured until confluence and then the medium was replaced with phenol red-free DMEM/F-12 supplemented with 10% dextran-coated charcoal stripped (DCS)-FCS, Antibiotic-Antimycotic 1×; Gibco), and 2 mmol/L L-alanyl-L-glutamine (GlutaMAX; Gibco). After 48 hours, ESCs were cultured in DCS-FCS supplemented with E 2 (10 −8 mol/L; Wako, Osaka, Japan) and medroxyprogesterone acetate (MPA; 10 −7 mol/L; Sigma-Aldrich) or ethanol as the vehicle control. The ESCs were treated with CSE at various concentrations (0.01%, 0.025%, 0.1, and 0.25%), or untreated, in addition to treatment with ovarian sex steroid hormones.
The culture medium, with or without CSE, was replaced every 3 days for up to 12 days. Experiments using ESCs from each patient were performed at least three times with different cell preparations.

| CSE preparations
CSE was prepared as previously described. 22 The cigarettes used in this study, Mevius (Japan Tobacco, Tokyo, Japan), contained 10 mg of tar and 0.8 mg of nicotine according to the manufacturer's report.
Using a constant airflow (0.3 L/min), the smoke of ten consecutive cigarettes was aspirated manually. The smoke was bubbled through 10 mL of phosphate-buffered saline (PBS) in a 50-mL polypropylene conical tube. The obtained CSE solution was defined as 100% (one cigarette per 1 mL) and then filtered through a 0.2-µm filter (ADVANTEC, Tokyo, Japan) to remove bacteria and large particles.
Fresh CSE solution was prepared prior to the start of each experiment. After diluting with PBS, the pH of the CSE was between 7.4 and 7.5 for each experiment.

| Cytotoxicity lactase dehydrogenase assay
The level of lactase dehydrogenase (LDH) released was determined using cytotoxicity LDH assay kit-WST (Dojindo, Kumamoto, Japan) to examine the effect of CSE treatment on cell viability. ESCs were seeded in 96-well plates at a density of 5 × 10 3 cells per well. After exposure to various concentrations of CSE for 24 hours, the working solution was subsequently added according to the manufacturer's instructions and the absorbance (optical density) at 490 nm was determined using a microplate reader (EnSpire; PerkinElmer, Inc). Each experiment was conducted in triplicate.

| Cell death detection via enzyme-linked immunosorbent assay
Apoptosis of ESCs was quantified by directly determining the ex-

| Semi-quantitative real-time polymerase chain reaction
Total RNA was isolated from cultured ESCs using the RNeasy Minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The first-strand cDNA synthesis kit ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan) was used for cDNA synthesis. α is the slope of the corresponding amplification plot. 23 For relative quantification, data were normalized against elongation factor-1α (EF-1α) as an internal control. The validated primer sequences are listed in Table 1.

| Western blot analysis
Whole cell lysates were prepared using lysis buffer containing mam-

| Determining VEGF and PRL levels using ELISA
The levels of VEGF and PRL released in cell culture supernatants were determined using a commercially available ELISA kit (Duoset ® ELISA kit; R&D Systems, Minneapolis, MN, USA). Intra-and interassay coefficients of variation in cell culture supernatants were 4.7% and 5.5% for VEGF and 2.2% and 8.9% for PRL, respectively.

| Morphological analysis of decidualized ESCs
For morphological assessments, each sample was stained using to quantitate the cell and nuclear shape changes associated with decidualization in 10 samples. 25 Cell SI was determined for low-power field (×100) images, whereas nuclear SI was determined for highpower field (×100) images.

| Statistical analysis
Data are presented as the mean ± standard deviation. Statistical analyses were performed using JMP 12 software. Groups were compared using one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons. Analysis of two independent factors was performed using the two-way ANOVA. The Tukey-Kramer test was used to compare the means between groups in which statistically significant differences were found. A value of P < .05 was considered statistically significant.

| Effects of CSE on cell proliferation, cytotoxicity, and apoptosis
To investigate the proliferative, cytotoxic, and apoptotic effects of CSE on ESCs, the cells were cultured with various concentrations of CSE for 24 hours. As shown in Figure 1A, up to 0.5% CSE had no effect on the cells; however, a significant dose-dependent inhibition of ESC proliferation was observed at treatment concentrations of >1% CSE. As shown in Figure 1B, up to 0.5% CSE had no cytotoxic effects whereas significant cytotoxicity was seen above 2%, and cytotoxicity trend observed at 1% CSE (P = .05). As shown in Figure 1C, apoptosis was observed at treatment concentrations above 1% CSE.
Therefore, for subsequent experiments, concentrations of CSE below 0.5% were used.

| Effect of angiogenic factors on mRNA expression
The effect of CSE on the mRNA expression of angiogenic factors was evaluated. As shown in Figure 2A, 0.01% CSE had no effect on VEGF mRNA expression compared to that in control cells, whereas a significant increase was observed at concentrations above 0.025%.
CSE did not affect SDF-1 mRNA expression at any of the CSE concentrations tested ( Figure 2B). As shown in Figure 2C, 0.25% CSE significantly decreased the expression of ANGPT1 mRNA, whereas no significant changes in ANPGT2 mRNA expression were observed at any concentration ( Figure 2D). Of the known angiogenic factors expressed in the human endometrium, the expression of only VEGF was significantly induced by CSE in a dose-dependent manner.

| Effects of CSE on the time course of VEGF secretion
To investigate the effects of CSE on the time course of VEGF secretion, ESCs were cultured with or without 0.25% CSE for various periods of time. A 0.25% concentration of CSE enhanced VEGF levels in a time-dependent manner ( Figure 3A,B). A 0.25% concentration of CSE caused a significant increase in VEGF production after 8 hours of culture compared with that in the control, and this increase continued until 12 days.

| Effect of CSE in HIF-1α protein and glucose transporter 1 mRNA expression
It has been demonstrated that VEGF mRNA expression increases as a downstream target gene of HIF-1α in ESCs. 26 To investigate the effect of CSE on HIF-1, ESCs were exposed to CSE at various concentrations under normoxic (20% O 2 ) or hypoxic (1% O 2 ) conditions for 4 hours. As shown in Figure 4A, CSE induced the accumulation of HIF-1α protein in a dose-dependent manner. The highest accumulation of HIF-1α protein was observed after treatment with 0.25% CSE, while the levels had attenuated with 0.5% CSE. These results suggest that CSE promotes the accumulation of HIF-1α to a similar extent as 1% O 2 . We then evaluated the mRNA expression of glucose transporter 1 (GLUT1), which is one of the important downstream genes of HIF-1α, as is VEGF. 27 Results showed a significant increase in the GLUT1 mRNA expression after culturing with concentrations above 0.025% CSE ( Figure 4B). These findings indicate that HIF-1α may be responsible for the CSE-induced VEGF and GLUT1 expression.

| Effects of CSE on decidualization
To investigate the effect of CSE on decidualization, the expression of PRL, which is a specific decidual marker, was analyzed. Cultured

| Effects of CSE on cell morphology
A defining feature of decidualization is the morphological change induced in ESCs, which includes the development of a round shape and cobblestone morphology with a concomitant increase in cell area. 25 To examine the effect of CSE on cell morphology, ESCs Data are presented as the mean ± SD, n = 4. Statistically significant differences are indicated as *P < .05, **P < .01 vs control (CSE 0%)

| Effects of CSE on mRNA levels of decidual specific factors
The effects of CSE on decidual specific factors, including IGFBP-1, HAND2, IL-15, and FGF9, were evaluated. ESCs treated with varying concentrations of CSE showed no significant differences in the mRNA expression levels of these factors (data not shown). Following the combined treatments of CSE together with E 2 and MPA, the IGFBP-1 mRNA expression levels significantly increased at 0.01% and 0.025% CSE concentrations compared to control. However, these levels were significantly suppressed at 0.25% CSE concentration, with a decreased trend in expression observed after treatment with 0.1% CSE (P = .07; Figure 8A). As shown in Figure 8B,C, exposure to E 2 and MPA resulted in significantly increased levels of HAND2 and IL-15 mRNA expression at 0.01% and 0.025% CSE concentrations, which were significantly suppressed at 0.1% and 0.25% CSE concentrations. These results revealed that differing concentrations of CSE exerted similar expression patterns for PRL and IGFBP-1. We also evaluated FGF9 expression, which is suppressed following exposure to E 2 and MPA, and is the downstream target of HAND2 in ESCs. 19 Our results showed that E 2 and MPA treatment attenuated FGF9 mRNA expression; however, no significant differences were noted, regardless of CSE concentration ( Figure 8D). The toxicity test for cigarette smoke is commonly used in animal or cell experiments, and CSE is widely employed in in vitro models. 28 Earlier studies report that 1% CSE approximately corresponds to exposures associated with smoking slightly less than two packs of cigarettes per day in pulmonary artery endothelial cells. 29,30 Based on these studies, we propose that 0.25% CSE concentration closely represents exposure faced by average smokers. However, whether CSE concentration is similar between pulmonary artery endothelial cells, which directly absorb cigarette smoke, and ESCs is unknown.

| D ISCUSS I ON
Concurring with our results, nicotine in cigarette smoke has been shown to induce VEGF expression in human ESCs, regardless of ovarian steroid hormones. 31 VEGF is essential for implantation and placentation 32 ; however, higher levels of VEGF may disrupt normal angiogenesis through an overstimulation of blood vessels leading to disturbed vascular architecture. 33,34 Taken together, it is important to consider the influence of these results of the present study on implantation, as the effects of CSE on angiogenesis may adversely affect the establishment of pregnancy.
In the promoters encoding VEGF genes, HIF-1α has been shown to directly bind to the hypoxia response element. In the human endometrium, HIF-1α is expressed with increasing intensity from the premenstrual to the menstrual phase. 35 Previous studies showed F I G U R E 5 Effects of cigarette smoke extract (CSE) on prolactin (PRL) mRNA expression and production. Human endometrial stromal cells (ESCs) were treated with CSE (0%, 0.01%, 0.025%, 0.1%, and 0.25%) and/or E 2 (10 −8 mol/L) + MPA (10 −7 mol/L) for up to 12 d. A,B, PRL mRNA expressions were assessed via quantitative real-time polymerase chain reaction and calculated after normalization to EF1α mRNA levels. C,D, PRL production in the culture supernatants treated with E 2 + MPA for 12 d was analyzed using enzyme-linked immunosorbent assay. Data are presented as the mean ± SD, n = 4. Statistically significant differences are indicated as *P < .05, **P < .01 vs control (CSE 0%) that both HIF-1α and HIF-2α have a functional role in embryo implantation. 36 GLUT1 has a HIF-1α binding sequence in its promoter. 27 In this study, CSE simultaneously increased GLUT1 mRNA expression and the accumulation of HIF-1α. We recently demonstrated that echinomycin, a small molecule inhibitor of HIF-1α activity, substantially reduced GLUT1 expression under hypoxia in ESCs, suggesting that HIF-1α plays a major role in regulating GLUT1 expression. The physiological role of GLUT1 is that in a hypoxic environment such as menstrual and implantation periods, ESCs increase extracellular glucose uptake and enhance glycolysis, thereby obtaining energy. 42 Herein, these findings suggest that CSE-induced HIF-1α plays a key role in the regulation of GLUT1 and VEGF expression.
The observation from this study that 0.25% CSE significantly decreased ANGPT1 expression, but had no effect on ANPGT2 expres-

F I G U R E 7
The shape index (SI) of circularity and cell area in endometrial stromal cells during decidualization with or without cigarette smoke extract (CSE) treatment. A, In order to quantify morphological differences, cellular and nuclear shape index were calculated from each condition in Figure 6 (n = 10). B, The cell areas were calculated from each condition in Figure 6 (n = 10). Each value represents mean ± SD; *P < .01 vs control (CSE 0% without E + MPA treatment) control, but expression levels were then suppressed at 0.1% and 0.25% CSE concentrations in the presence of E 2 and MPA. However, without E 2 and MPA, CSE had no effect on the levels of PRL expression, suggesting that CSE affects PRL levels during decidualization.
The observed morphological changes correlated with the changes in PRL levels after exposure to different CSE concentrations. Cadmium, one of the major contaminants of cigarette smoke, markedly elevates PRL levels and stimulates decidualization in ESCs. 44 Another study also showed that CSE increased the expression of endometrial homeobox 10 and the progesterone receptor and promoted early decidualization in immortalized endometrial cell lines of ESC. 45 In this study, treatment with 0.01% and 0.025% CSE also upregulated IGFBP-1 mRNA in ESCs. The time point of IGFBP-1 secretion, which is about 10 d after the luteinizing hormone peak in vivo, is relevant for a marked reduction in endometrial receptivity and a rapidly increasing risk of implantation failure. 8 Therefore, CSE may affect the endometrial receptivity by upregulating IGFBP-1.
There are very few reports that CSE suppresses decidualization.
To the best of our knowledge, there is one report using female rat models showing the potential effects of nicotine on endometrial decidualization by assessing by the weight of the uterus after mechanically induced decidualization. The authors concluded that there was an adverse effect on the decidualization process resulting in a lower uterus weight after nicotine administration. 46 In the present study, we showed that CSE resulted in differential gene expressions depending on the concentration tested. This may be attributed to con- were treated with CSE (0%, 0.01%, 0.025%, 0.1%, and 0.25%) and/or E 2 (10 −8 mol/L) + MPA (10 −7 mol/L) for up to 12 d. The mRNA expression of (A) insulin-like growth factor-binding protein 1 (IGFBP-1), (B) heart and neural crest derivatives-expressed transcript 2 (HAND2), (C) interleukin 15 (IL-15), and (D) fibroblast growth factor 9 (FGF9) were analyzed via RT-PCR. Data were normalized to the EF-1α housekeeping gene and are shown as the mean ± SD (n = 4). *P < .05, **P < .01 vs control (CSE 0%) differential gene expressions depending on the concentration tested and also lead to the development of therapeutic strategies.
Collectively, our study demonstrates that CSE above 0.025% enhances the expression of angiogenic factors, and CSE above 0.01% affects the expression of decidual specific factors in ESCs in the presence of E 2 and MPA. These results highlight that exposure to even a small amount of cigarette smoke could affect ESCs. Our study provides a novel insight in that cigarette smoke directly affects the human endometrium. Moreover, our results support epidemiological studies that cigarette smoke has an adverse effect on reproductive outcome.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

H U M A N R I G HT S , I N FO R M E D CO N S E NT, A N D E TH I C A L A PPROVA L
All the procedures were followed in accordance with the ethical standards of the institutional ethical committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. In this study, informed consent was obtained from all the patients who underwent hysterectomy. This study was approved by the Institutional Review Board at Kansai Medical University.

A N I M A L S TU D I E S
This article does not contain any studies with human and animal subjects performed by the any of the authors.