Role of chemerin/CMKLR1 in the maintenance of early pregnancy

Abstract

Chemerin is a cytokine that attracts much attention in the reproductive process. This study aimed to explore the effects of chemerin and its receptor chemokine-like receptor 1 (CMKLR1) on the maintenance of early pregnancy. The expression levels of chemerin and CMKLR1 in the decidua tissues of 20 early normal pregnant women and 20 early spontaneous abortion women were examined by Western blot and real-time polymerase chain reaction analyses. CMKLR1 receptor antagonist (α-NETA) was then intrauterinely injected into normal pregnant mice model to assess its effect on the outcome of pregnancy and the phosphorylation rate of ERK1/2 in decidua tissues. We found that the expression level of chemerin in women who had experienced early spontaneous abortion was lower than in those who had experienced normal early pregnancy (P<0.01); conversely, CMKLR1 expression was higher in the former than in the latter (P<0.01). In a pregnant-mouse model, the embryo resorption rate of α-NETA group was higher than that in the negative control group (61.5% vs. 10.8%) (P<0.001). Compared with the control group, ERK1/2 phosphorylation in decidua tissues decreased in the α-NETA-treated group (P<0.01). These results suggested that the inhibition of the chemerin/CMKLR1 signaling pathway can lead to the abortion of mouse embryos, and that chemerin/CMKLR1 may play an important role in the maintenance of early pregnancy possibly by regulating ERK1/2 phosphorylation.

Keywords

chemerin / CMKLR1 / early abortion

Introduction

Spontaneous abortion is common and occurs in approximately 10%–15% of normal couples, among which 2%–3% is recurrent abortion. More than 80% of spontaneous abortion occurs in early pregnancy (within 12 weeks of pregnancy) [¹,²]. Early spontaneous abortion is attributable to multiple causes, such as embryonic chromosome or gene abnormalities [³–⁶], endocrine abnormalities [⁷], maternal anatomical abnormalities [⁸,⁹], immune abnormalities [¹⁰–¹³], local thrombosis [¹⁴], and so on.

In recent years, researchers have focused on the relation between immunization and early abortion. Chemerin is a cytokine that plays a role in the development of inflammation [¹⁵], insulin resistance [¹⁶–¹⁸], and cardiovascular disease [¹⁹–²¹]. It is also a chemotactic factor for innate immune cells and a critical player in the initiation of immune responses. Chemokine-like receptor 1 (CMKLR1) is a kind of G protein coupled receptor and is also a natural ligand of chemerin [²²–²⁴]. Many studies showed that chemerin/CMKLR1 was closely related to the female reproductive process. Chemerin expression levels in the peripheral blood of pregnant women were significantly higher than non-pregnant women [²⁵], and chemerin content in umbilical cord blood of older women was remarkably higher than normal aged pregnant women [²⁶]. Both chemerin and CMKLR1 were expressed in placental trophoblast cells and a variety of cells involved in the uterus decidualization [²⁷–³⁰]. Chemerin was highly expressed in uterine stromal cells and extravillous trophoblast cells of women during early pregnancy [²⁸]. Chemerin combined with CMKLR1 can induce the peripheral blood dendritic cells, whereas dendritic cells in the peripheral blood of women undergoing the fetal growth restriction were significantly lower than normal pregnant women and non-pregnant women [³¹]. Except that, Claudia Carlino et al. demonstrated that chemerin is upregulated during decidualization and contributes to NK cell accumulation and vascular remodeling during early pregnancy [²⁸].

Therefore, we hypothesized that chemerin/CMKLR1 might play an important role in the maintenance of early pregnancy and prevention of spontaneous abortion caused by abnormal immunity. To explore this hypothesis in the present study, we collected the decidua tissues of early normal pregnant women and spontaneous abortion women and examined their chemerin and CMKLR1 expression levels. Then, we used Kunming mice to construct pregnant mice model and treated them with CMKLR1 receptor antagonist (α-NETA) or not. We evaluated the embryo resorption rate and phosphorylation of ERK1/2 in decidua tissues of different groups. Our results suggest that chemerin/CMKLR1 signaling pathway plays an important role in the maintenance of early pregnancy and it functions possibly through the regulation of phosphorylation of ERK1/2.

Materials and methods

Materials and agents

Antibodies against chemerin and CMKLR1 were supplied by Abcam (Cambridge, MA, UK). The antibody against Phos ERK1/2 was purchased from ABclonal. CMKLR1 inhibitor (α-NETA) was purchased from Santa Cruz (USA). Total RNA kit was supplied by Invitrogen (USA). 5 × PrimeScript^TM RT Master Mix and SYBR® Premix Ex Taq^TM were purchased from TaKaRa (Japan).

Decidua tissue sample

Decidua tissues of 40 pregnant women treated with complete curettage of uterine cavity were collected from November 2014 to March 2015 at Xiangyang Central Hospital. This study was approved by the Ethics Committee of Xiangyang Central Hospital in accordance with the Declaration of Helsinki. All the patients above were informed of this research and volunteered to donate their decidua tissues for research. All the participants were 20–35 years old, and the gestational age was around 6 to 8 weeks. Twenty of these patients have a history of at least two spontaneous abortions and the ultrasound confirmed that the fetal heart beat was no longer detectable, while the other 20 participants who were undergoing artificial miscarriage (selective abortion) had no history of miscarriage and the ultrasound confirmed that the fetal samples had normal heart beat. No significant differences were observed in the mean age and gestational age between the two groups. Immune diseases such as anti-phospholipid syndrome and systemic lupus erythematous treated with immunosuppressive agents and patients suffering from infectious diseases in three months were considered as exclusion criteria. Furthermore, chromosomes of the couples were normal and the miscarriage was not caused by the male. All aborted fetuses excluded the chromosomes or genetic abnormalities by gene analysis.

Animals

In this study, eight-week-old female Kunming mice were supplied by Animal Center of Huazhong University of Science and Technology and were used to establish a pregnancy model. All animals were housed under a 12 h dark-light cycle at 22±1 °C and had free access to food and water ad libitum. The experimental procedures of this study were approved by the Ethics Committee of Huazhong University of Science and Technology.

Superovulation

The female mice were injected intraperitoneally with 10 IU of HMG followed by injection with another 10 IU of HMG 24 h later. Then, the mice were intraperitoneally administered with 10 IU of hCG for another 24 h later. After injection of hCG, female Kunming mice were mated with male Kunming mice and checked for a vaginal plug next morning. Presence of a vaginal plug was considered to indicate D1 of pregnancy.

Animal treatment and sample collection

The normal pregnant mice were randomly divided into three groups equally: one treatment group and two control groups. The control groups were subdivided to negative control group and sham control group. The mice were entered into study on the day of diestrus using vaginal smears. All the left uterine horn of the pregnant mice in three groups accepted treatment at D6, D9, and D12, respectively. While as a self-control, all the right uterine horn of the pregnant mice received no treatment of any kind. In the treatment group and negative control group, the Kunming mice received an injection of α-NETA (5 µg/20 µL) and 20 µL of PBS, respectively, whereas the Kunming mice in sham control group were injected with nothing. On D14, the mice were sacrificed and the uteri were removed. Then, morphology changes in the uteri were observed and the total number of implantation and resorption sites was recorded. The percentage of abortions was calculated as the ratio of resorption sites to the total number of implantation sites (resorption plus normal implantation sites).

Quantitative real-time polymerase chain reaction (PCR) analysis

Total RNA was extracted from decidua tissues using Trizol reagent (Invitrogen, USA). Concentrations and purities of RNA were estimated using a D30 spectrophotometer (Eppendorf, Germany). If the ratio of the absorbance at 260 nm to the absorbance at 280 nm was between 1.8 and 2.0, then the RNA was considered to be relatively pure and converted into first strand cDNA using 5 × PrimeScript^TM RT Master Mix (Takara, Japan). Then, the quantitative real-time PCR was performed with SYBR® Premix Ex Taq^TM (Takara, Japan) on a Light Cycler®96(Roche, USA). The primer sequence (Invitrogen, Shanghai, China) is shown in Table 1. The relative mRNA expression level was calculated by 2^−ΔΔCt method.

Western blot

Western blot analysis was used to determine the expression levels of chemerin and CMKLR1 in the decidua tissues from the study participants and the expression level of phosphorylated ERK1/2 in decidua tissues from the mice model. Total protein was extracted from decidua sample. Protein concentrations were determined by a nucleic acid protein detector (BioTek, USA) and adjusted to 20 µg/µL. Forty µg of each protein sample was resolved on a 12% SDS-PAGE. Then, proteins were transferred to PVDF membranes and incubated with antibodies specific to chemerin (1:500 dilution), CMKLR1 (1:750 dilution), phosphorylated ERK1/2 (1:750 dilution) and GAPDH (1:4000 dilution). Subsequently, the membranes were incubated with appropriate secondary antibody (1:4000 dilution) and visualized using Odyssey imaging system (LI-COR, USA). Relative protein expression levels were analyzed by Image J software.

Statistical analysis

The collected data were analyzed using SPSS 19.0. The relative expression levels of chemerin and CMKLR1 between early spontaneous abortion women and normal early pregnancy women were determined by Student t-test. The embryo resorption rate was evaluated by Chi-square test when the number was above 5. Otherwise, the Fisher’s exact test was applied. P<0.05 was considered as statistically significant.

Results

The expression of chemerin and CMKLR1 between early spontaneous abortion women and normal early pregnancy women has a significant difference

To determine possible roles of chemerin and CMKLR1 in early spontaneous abortion and early pregnancy, we examined the expression level of chemerin and CMKLR1 in the decidua tissues of 20 early spontaneous abortion women and 20 normal early pregnancy women by real-time PCR and Western blot. Our results showed that the expression of chemerin in early spontaneous abortion women was significantly lower than that in normal early pregnancy women (P<0.01; Fig. 1A, 1B, and 1E). However, the expression of CMKLR 1 in early spontaneous abortion women was significantly higher than that in normal early pregnancy women (P<0.05; Fig. 1C, 1D, and 1E).

Antagonist of CMKLR1 receptor decreases the continued pregnancy rate of mice

To address the role of chemerin/CMKLR1 in continued pregnancy, α-NETA, the antagonist of CMKLR1 receptor was injected into the left uterine horn of the pregnant female mice. After the mice were sacrificed on D14, the uteri were removed and the total number of implantation and resorption sites was recorded. The percentage of abortions was calculated as the ratio of resorption sites to the total number of implantation sites (resorption plus normal implantation sites), as described previously. The macroscopic observations of the embryos attached to the uterus on D14 showed that the treatment of pregnant Kunming mice with α-NETA in early pregnancy significantly increased the abortion rate compared with the negative control group (61.5% vs. 10.8%, P<0.001; Fig. 2A and 2B). No significant difference was observed in the abortion rate between negative control group and sham group (10.8% vs. 11.1%, P>0.05; Fig. 2A and 2B).

Antagonist of CMKLR1 receptor decreases the phosphorylation rate of ERK1/2 in decidua tissues

After the pregnant female Kunming mice were sacrificed on D14, the decidua tissues were collected to test the phosphorylation of ERK1/2 by Western blot. The phosphorylation level of ERK1/2 in the α-NETA-injected group decreased significantly compared with negative control group and sham control group (P<0.01; Fig. 3A and 3B).

Discussion

As a semi-allograft to the mother, embryo will be denied by the mother’s body if an imbalance occurs in immunological tolerance [³²]. During the key events of pregnancy, such as blastocyst implantation, immunological tolerance, and trophoblast invasion, alterations in the immune response occurred and the decidua of the uterus is importantly involved in these alterations through changes in cytokine expression and leukocyte cell recruitment [³³,³⁴]. Chemerin is a cytokine that acts as chemoattractant for innate immune cells and has been suggested as an important player in the initiation of immune responses. Chemerin was highly expressed in uterine stromal cells and extravillous trophoblast cells during early pregnancy [²⁸]. Moreover, uterine stromal cells from early pregnant women exhibited and released higher levels of chemerin compared with uterine stromal cells from non-pregnant women [²⁸]. These findings strongly supported the idea that chemerin was closely related to the pregnancy. However, the possible roles of chemerin in the maintenance of pregnancy have rarely been investigated.

To determine a possible role of chemerin in early pregnancy, the decidua tissues of 20 early spontaneous abortion women and 20 normal early pregnant women treated with complete curettage of uterine cavity were collected from November 2014 to March 2015 at Xiangyang Central Hospital and the expression of chemerin and CMKLR1 in decidua tissues of early pregnant women were confirmed by real-time PCR and Western blot. In general, real-time PCR detection of mRNA for chemerin and CMKLR1 was consistent with the protein results. Our experiments showed that chemerin in decidua tissues of early spontaneous abortion women were significantly lower than that in normal pregnant women both at mRNA and protein levels (Fig. 1A, 1B, and 1E). These results can directly confirm that chemerin/CMKLR1 system was involved in the process of normal pregnancy. CMKLR1 was high in decidua tissues of spontaneous abortion women at both mRNA and protein levels (Fig. 1C, 1D, and 1E). Taken together, these findings further support that chemerin/CMKLR1 may play an important role in the continued pregnancy process and chemerin may be a new potential diagnostic biomarker for successful pregnancy.

Intricate interplay between different cell types was involved in reproduction process, thus it was too complex to adequately study with cell culture approaches. Consequently, it prompted us to explore the possible effects of chemerin and CMKLR1 on the continued pregnancy by a direct in vivo study. We established pregnant mice model and injected α-NETA, which was the antagonist of CMKLR1 receptor, into the left uterine horn of mice at D6, D9, and D12. Our results showed that the embryo absorption rate of pregnant mice increased significantly after treatment with α-NETA (Fig. 2A and 2B). This result indicated that the inhibition of chemerin/CMKLR1 reduced the continued pregnancy rate and increased the abortion rate in mice.

MAPK/ERK1/2 signaling pathway was associated with pregnancy loss [³⁵–³⁹]. Masanori et al. demonstrated that inactivation of the MAPK pathway by Listeriamonocytogenes can cause infectious abortion [³⁵]. Meanwhile, MAPK cascade is a key signaling pathway, which is recruited by activated CMKLR1 receptor. Several reports have demonstrated that CMKLR1 activates MEK1, which in turn phosphorylates ERK1/2. To confirm whether chemerin maintains the early pregnancy through MAPK/ERK1/2 signaling pathway, the decidua tissues of the pregnant Kunming mice were collected and the phosphorylation level of ERK1/2 was determined. Western blot analysis showed that treatment with α-NETA decreased the phosphorylation of ERK1/2 (Fig. 3A and 3B). This result suggested that chemerin/CMKLR1 acted as a protector in the maintenance of early pregnancy possibly through phosphorylation of ERK1/2.

The mechanisms underlying pregnancy failure were complex and most of them remain unknown [⁴⁰]. Consequently, inactivation of the chemerin/CMKLR1 signaling pathway might only be part of these mechanisms. In addition, chemerin playing a role in the maintenance of early pregnancy might not only be through phosphorylation of ERK1/2. Thus, the exact signaling pathways need to be further evaluated in vitro and in vivo.

In summary, our data demonstrate the expression of chemerin and CMKLR1 in human deciduous tissues and provide the first evidence of the direct action of chemerin/CMKLR1 in the maintenance of pregnancy. Together with the suppressive effects of antagonist of CMKLR1 receptor on continuing pregnancy, the results of the present study suggest that chemerin/CMKLR1 is one of the regulators of continued pregnancy. Therefore, further studies about molecular mechanism of chemerin/CMKLR1 in maintaining early pregnancy will aid in the prevention of abortion and provide potential contraception in the future.

References

[1] Rai R, Regan L. Recurrent miscarriage. Lancet 2006; 368(9535): 601–611

[2] Saravelos SH, Cocksedge KA, Li TC. The pattern of pregnancy loss in women with congenital uterine anomalies and recurrent miscarriage. Reprod Biomed Online 2010; 20(3): 416–422

[3] Branch DW, Gibson M, Silver RM. Recurrent miscarriage. N Engl J Med 2010; 363(18): 1740–1747

[4] Sugiura-Ogasawara M, Aoki K, Fujii T, Fujita T, Kawaguchi R, Maruyama T, Ozawa N, Sugi T, Takeshita T, Saito S. Subsequent pregnancy outcomes in recurrent miscarriage patients with a paternal or maternal carrier of a structural chromosome rearrangement. J Hum Genet 2008; 53(7): 622–628

[5] Su MT, Lin SH, Chen YC. Genetic association studies of angiogenesis- and vasoconstriction-related genes in women with recurrent pregnancy loss: a systematic review and meta-analysis. Hum Reprod Update 2011; 17(6): 803–812

[6] Franssen MT, Musters AM, van der Veen F, Repping S, Leschot NJ, Bossuyt PM, Goddijn M, Korevaar JC. Reproductive outcome after PGD in couples with recurrent miscarriage carrying a structural chromosome abnormality: a systematic review. Hum Reprod Update 2011; 17(4): 467–475

[7] Arredondo F, Noble LS. Endocrinology of recurrent pregnancy loss. Semin Reprod Med 2006; 24(1): 33–39

[8] Saravelos SH, Cocksedge KA, Li TC. Prevalence and diagnosis of congenital uterine anomalies in women with reproductive failure: a critical appraisal. Hum Reprod Update 2008; 14(5): 415–429

[9] Saravelos SH, Yan J, Rehmani H, Li TC. The prevalence and impact of fibroids and their treatment on the outcome of pregnancy in women with recurrent miscarriage. Hum Reprod 2011; 26(12): 3274–3279

[10] Alijotas-Reig J, Ferrer-Oliveras R; EUROAPS Study Group. The European Registry on Obstetric Antiphospholipid Syndrome (EUROAPS): a preliminary first year report. Lupus 2012; 21(7): 766–768

[11] Danza A, Ruiz-Irastorza G, Khamashta M. Antiphospohlipid syndrome in obstetrics. Best Pract Res Clin Obstet Gynaecol 2012; 26(1): 65–76

[12] Rowe JH, Ertelt JM, Xin L, Way SS. Pregnancy imprints regulatory memory that sustains anergy to fetal antigen. Nature 2012; 490(7418): 102–106

[13] Tang AW, Alfirevic Z, Quenby S. Natural killer cells and pregnancy outcomes in women with recurrent miscarriage and infertility: a systematic review. Hum Reprod 2011; 26(8): 1971–1980

[14] Davenport WB, Kutteh WH. Inherited thrombophilias and adverse pregnancy outcomes: a review of screening patterns and recommendations. Obstet Gynecol Clin North Am 2014; 41(1): 133–144

[15] Ernst MC, Sinal CJ. Chemerin: at the crossroads of inflammation and obesity. Trends Endocrinol Metab 2010; 21(11): 660–667

[16] Corona-Meraz FI, Navarro-Hernandez RE, Ruiz-Quezada SL, Madrigal-Ruiz PM, Castro-Albarran J, Chavarria-Avila E, Guzman-Ornelas MO, Gomez-Banuelos E, Petri MH, Ramirez-Cedano JI, Aguilar-Aldrete ME, Rios-Ibarra C, Vazquez-Del Mercado M. Inverse relationship of the CMKLR1 relative expression and chemerin serum levels in obesity with dysmetabolic phenotype and insulin resistance. Mediators Inflamm 2016; 2016:3085390

[17] Becker M, Rabe K, Lebherz C, Zugwurst J, Göke B, Parhofer KG, Lehrke M, Broedl UC. Expression of human chemerin induces insulin resistance in the skeletal muscle but does not affect weight, lipid levels, and atherosclerosis in LDL receptor knockout mice on high-fat diet. Diabetes 2010; 59(11): 2898–2903

[18] Sell H, Laurencikiene J, Taube A, Eckardt K, Cramer A, Horrighs A, Arner P, Eckel J. Chemerin is a novel adipocyte-derived factor inducing insulin resistance in primary human skeletal muscle cells. Diabetes 2009; 58(12): 2731–2740

[19] Kennedy AJ, Yang P, Read C, Kuc RE, Yang L, Taylor EJ, Taylor CW, Maguire JJ, Davenport AP. Chemerin elicits potent constrictor actions via chemokine-like receptor 1 (CMKLR1), not G-protein-coupled receptor 1 (GPR1), in human and rat vasculature. J Am Heart Assoc 2016; 5(10): e004421

[20] Ferland DJ, Watts SW. Chemerin: a comprehensive review elucidating the need for cardiovascular research. Pharmacol Res 2015; 99: 351–361

[21] Dessein PH, Tsang L, Woodiwiss AJ, Norton GR, Solomon A. Circulating concentrations of the novel adipokine chemerin are associated with cardiovascular disease risk in rheumatoid arthritis. J Rheumatol 2014; 41(9): 1746–1754

[22] Samson M, Edinger AL, Stordeur P, Rucker J, Verhasselt V, Sharron M, Govaerts C, Mollereau C, Vassart G, Doms RW, Parmentier M. ChemR23, a putative chemoattractant receptor, is expressed in monocyte-derived dendritic cells and macrophages and is a coreceptor for SIV and some primary HIV-1 strains. Eur J Immunol 1998; 28(5): 1689–1700

[23] Parolini S, Santoro A, Marcenaro E, Luini W, Massardi L, Facchetti F, Communi D, Parmentier M, Majorana A, Sironi M, Tabellini G, Moretta A, Sozzani S. The role of chemerin in the colocalization of NK and dendritic cell subsets into inflamed tissues. Blood 2007; 109(9): 3625–3632

[24] Luangsay S, Wittamer V, Bondue B, De Henau O, Rouger L, Brait M, Franssen JD, de Nadai P, Huaux F, Parmentier M. Mouse ChemR23 is expressed in dendritic cell subsets and macrophages, and mediates an anti-inflammatory activity of chemerin in a lung disease model. J Immunol 2009; 183(10): 6489–6499

[25] Garces MF, Sanchez E, Ruíz-Parra AI, Rubio-Romero JA, Angel-Müller E, Suarez MA, Bohórquez LF, Bravo SB, Nogueiras R, Diéguez C, Caminos JE. Serum chemerin levels during normal human pregnancy. Peptides 2013; 42: 138–143

[26] Boutsikou T, Briana DD, Boutsikou M, Kafalidis G, Stamati L, Baka S, Hassiakos D, Gourgiotis D, Malamitsi-Puchner A. Cord blood chemerin and obestatin levels in large for gestational age infants. J Matern Fetal Neonatal Med 2013; 26(2): 123–126

[27] Garces MF, Sanchez E, Acosta BJ, Angel E, Ruíz AI, Rubio-Romero JA, Diéguez C, Nogueiras R, Caminos JE. Expression and regulation of chemerin during rat pregnancy. Placenta 2012; 33(5): 373–378

[28] Carlino C, Trotta E, Stabile H, Morrone S, Bulla R, Soriani A, Iannitto ML, Agostinis C, Mocci C, Minozzi M, Aragona C, Perniola G, Tedesco F, Sozzani S, Santoni A, Gismondi A. Chemerin regulates NK cell accumulation and endothelial cell morphogenesis in the decidua during early pregnancy. J Clin Endocrinol Metab 2012; 97(10): 3603–3612

[29] Duan DM, Niu JM, Lei Q, Lin XH, Chen X. Serum levels of the adipokine chemerin in preeclampsia. J Perinat Med 2011; 40(2): 121–127

[30] Wang Q, Leader A, Tsang BK. Inhibitory roles of prohibitin and chemerin in FSH-induced rat granulosa cell steroidogenesis. Endocrinology 2013; 154(2): 956–967

[31] Lee KJ, Shim SH, Kang KM, Kang JH, Park DY, Kim SH, Farina A, Shim SS, Cha DH. Global gene expression changes induced in the human placenta during labor. Placenta 2010; 31(8): 698–704

[32] Trowsdale J, Betz AG. Mother’s little helpers: mechanisms of maternal-fetal tolerance. Nat Immunol 2006; 7(3): 241–246

[33] Kyurkchiev D, Ivanova-Todorova E, Hayrabedyan S, Altankova I, Kyurkchiev S. Female sex steroid hormones modify some regulatory properties of monocyte-derived dendritic cells. Am J Reprod Immunol 2007; 58(5): 425–433

[34] Ivanova E, Kyurkchiev D, Altankova I, Dimitrov J, Binakova E, Kyurkchiev S. CD83 monocyte-derived dendritic cells are present in human decidua and progesterone induces their differentiation in vitro. Am J Reprod Immunol 2005; 53(4): 199–205

[35] Hashino M, Tachibana M, Nishida T, Hara H, Tsuchiya K, Mitsuyama M, Watanabe K, Shimizu T, Watarai M. Inactivation of the MAPK signaling pathway by Listeria monocytogenes infection promotes trophoblast giant cell death. Front Microbiol 2015; 6: 1145

[36] Wang S, Cao C, Piao H, Li Y, Tao Y, Zhang X, Zhang D, Sun C, Zhu R, Wang Y, Yuan M, Li D, Du M. Tim-3 protects decidual stromal cells from toll-like receptor-mediated apoptosis and inflammatory reactions and promotes Th2 bias at the maternal-fetal interface. Sci Rep 2015; 5(1): 9013

[37] Hu WT, Li MQ, Liu W, Jin LP, Li DJ, Zhu XY. IL-33 enhances proliferation and invasiveness of decidual stromal cells by up-regulation of CCL2/CCR2 via NF-kB and ERK1/2 signaling. Mol Hum Reprod 2014; 20(4): 358–372

[38] Zhu R, Huang YH, Tao Y, Wang SC, Sun Ch, Piao HL, Wang XQ, Du MR, Li DJ. Hyaluronan up-regulates growth and invasion of trophoblasts in an autocrine manner via PI3K/AKT and MAPK/ERK1/2 pathways in early human pregnancy. Placenta 2013; 34(9): 784–791

[39] Xie KM, Hou XF, Li MQ, Li DJ. NME1 at the human maternal-fetal interface downregulates titin expression and invasiveness of trophoblast cells via MAPK pathway in early pregnancy. Reproduction 2010; 139(4): 799–808

[40] Laird SM, Tuckerman EM, Cork BA, Linjawi S, Blakemore AI, Li TC. A review of immune cells and molecules in women with recurrent miscarriage. Hum Reprod Update 2003; 9(2): 163–174

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature