Interimplantation Sites Research Articles

FOXO3a is essential for murine endometrial decidualization through cell apoptosis during early pregnancy.

Embryo implantation is essential for normal pregnancy, and the process of decidualization is critical for embryo implantation. However, the mechanism of decidualization during early pregnancy is still unknown. Forkhead box O3a (FOXO3a) is the most important functional transcription factor of the forkhead box family and is a highly conserved transcription factor of apoptosis-related genes. In the mouse uterus, FOXO3a was found to be expressed regularly from Days 1-7 of early pregnancy. Upon further exploration, it was found that FOXO3a was expressed at significantly higher levels at the implantation site than at the interimplantation site on Days 5-7 of pregnancy. Under artificial decidualization, FOXO3a was highly expressed in the first and second decidual zones. After decidualization, the expression of FOXO3a was significantly increased both in vivo and vitro. In primary stromal cells, apoptosis was reduced by decreased expression of FOXO3a after inducing decidualization. Moreover, when FOXO3a-small interfering RNA was transfected into the uteri of mice, the expression of decidualization- and apoptosis-related factors was impaired. Thus, FOXO3a might play an important role in decidualization during early pregnancy, and cell apoptosis might be one of pathways for FOXO3a-regulated decidualization.

Expression and function of Pdcd4 in mouse endometrium during early pregnancy.

Embryo implantation is a complex process involving synchronised crosstalk between a receptive endometrium and functional blastocysts. Apoptosis plays an important role in this process as well as in the maintenance of pregnancy. In this study, we analysed the expression pattern of programmed cell death 4 (Pdcd4), a gene associated with apoptosis in the mouse endometrium, during early pregnancy and pseudopregnancy by real-time quantitative polymerase chain reaction, in situ hybridisation, Western blotting and immunohistochemistry. The results showed that Pdcd4 was increased along with days of pregnancy and significantly reduced at implantation sites (IS) from day 5 of pregnancy (D5). The level of Pdcd4 at IS was substantially lower than that at interimplantation sites (IIS) on D6 and D7. In addition, Pdcd4 expression in the endometrium was reduced in response to artificially induced decidualisation in vivo and in vitro Downregulation of Pdcd4 gene expression in cultured primary stromal cells promoted decidualisation, while upregulation inhibited the decidualisation process by increasing apoptosis. These results demonstrate that Pdcd4 is involved in stromal cell decidualisation by mediating apoptosis and therefore plays a role in embryo implantation in mice.

Comparative Transcriptomic Analysis of Embryo Implantation in Mice and Rats

Background/Aims: Embryo implantation is an essential process for eutherian pregnancy, but this process varies across eutherians. The genomic mechanisms that led to the emergence and diversification of embryo implantation are largely unknown. Methods: In this study, we analyzed transcriptomic changes during embryo implantation in mice and rats by using RNA-seq. Bioinformatics and evolutionary analyses were performed to characterize implantation-associated genes in these two species. Results: We identified a total of 518 differentially expressed genes in mouse uterus during implantation, of which 253 genes were up-regulated and 265 genes were down-regulated at the implantation sites compared with the inter-implantation sites. In rat uterus, there were 374 differentially expressed genes, of which 284 genes were up-regulated and 90 genes were down-regulated. A cross-species comparison revealed that 92 up-regulated genes and 20 down-regulated genes were shared. The differences and similarities between mice and rats were investigated further at the gene ontology, pathway, network, and causal transcription factor levels. Additionally, we found that embryo implantation might have evolved through the recruitment of ancient genes into uterine expression. The evolutionary rates of the differentially expressed genes in mouse and rat uterus were significantly lower than those of the non-changed genes, indicating that implantation-related genes are evolutionary conserved due to high selection pressure. Conclusion: Our study provides insights into the molecular mechanisms involved in the evolution of embryo implantation.

Assessment of Embryo-Induced Transcriptomic Changes in Hamster Uterus Using RNA-Seq

Background/Aims: The mouse is widely used as an animal model for studying human embryo implantation. However, the mouse is unique in that both ovarian progesterone and estrogen are critical to implantation, whereas in the majority of species (e.g. human and hamster) implantation can occur in the presence of progesterone alone. Methods: In this study, we analyzed embryo-induced transcriptomic changes in the hamster uterus during embryo implantation by using RNA-seq. Differentially expressed genes were characterized by bioinformatic analysis. Results: We identified a total of 781 differentially expressed genes, of which 367 genes were up-regulated and 414 genes were down-regulated at the implantation site compared to the inter-implantation site. Functional clustering and gene network analysis highlighted the cell cycle process in uterus upon embryo implantation. By examining of the promoter regions of differentially expressed genes, we identified 7 causal transcription factors. Additionally, through connectivity map (CMap) analysis, multiple compounds were identified to have potential anti-implantation effects due to their ability to reverse embryo-induced transcriptomic changes. Conclusion: Our study provides a valuable resource for in-depth understanding of the mechanism underlying embryo implantation.

Roles of DEK in the endometrium of mice in early pregnancy

Embryo implantation is a complex process requiring reciprocal interactions between implantation-competent blastocysts and receptive uteri. Accumulating evidence from Digital Protein Expression Profiling indicates that DEK protein expression at implantation sites (ISs) was much higher than that at inter-implantation sites (IISs). In this study, we investigated the expression of DEK in mouse uterus by immunohistochemistry (IHC), Western blotting. We explored its function during decidualization of uterine stromal cells by inhibiting the expression of DEK. In further study of mechanism, the cell proliferation, apoptosis and DNA damage were detected after inhibiting DEK during decidualization of stromal cells. The results suggest that DEK participates in decidualization of stromal cells through mediating cell proliferation, apoptosis and DNA repair.

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Genome-wide analysis and functional prediction of long non-coding RNAs in mouse uterus during the implantation window.

Establishment of the receptive uterus is a crucial step for embryo implantation. In this study, the expression profiles and characterization of long non-coding RNAs (lncRNAs) in pregnant mouse uteri on day 4, day 5 at implantation sites and inter-implantation sites were conducted using RNA-seq. A total of 7,764 putative lncRNA transcripts were identified, including 6,179 known lncRNA transcripts and 1,585 novel lncRNA transcripts. Bioinformatics analysis of the cis and trans lncRNA targets showed that the differentially expressed lncRNAs were mainly involved in tissue remodelling, immune response and metabolism-related processes, indicating that lncRNAs could be involved in the regulation of embryo implantation. We also discovered that differentially expressed lncRNAs might regulate multiple signalling pathways that play an important role in the regulation of embryo implantation. In addition, nine known lncRNAs and four novel lncRNAs were randomly selected and validated by qRT-PCR. The expression of Tug1, Neat1, Gas5, Malat1, H19 and Rmst were significantly regulated in the mouse uterus during the implantation window. Our results are the first to systematically identify lncRNAs in the mouse uterus and provide a catalogue of lncRNAs for further understanding their functions in pregnant mouse uteri during the implantation window.

HB-EGF regulates Prss56 expression during mouse decidualization via EGFR/ERK/EGR2 signaling pathway.

Embryo implantation and decidualization are key steps for successful reproduction. Although numerous factors have been identified to be involved in embryo implantation and decidualization, the mechanisms underlying these processes are still unclear. Based on our preliminary data, Prss56, a trypsin-like serine protease, is strongly expressed at implantation site in mouse uterus. However, the expression, regulation and function of Prss56 during early pregnancy are still unknown. In mouse uterus, Prss56 is strongly expressed in the subluminal stromal cells at implantation site on day 5 of pregnancy compared to inter-implantation site. Under delayed implantation, Prss56 expression is undetected. After delayed implantation is activated by estrogen, Prss56 is obviously induced at implantation site. Under artificial decidualization, Prss56 signal is seen at the primary decidual zone at the initial stage of artificial decidualization. When stromal cells are induced for in vitro decidualization, Prss56 expression is significantly elevated. Dtprp expression under in vitro decidualization is suppressed by Prss56 siRNA. In cultured stromal cells, HB-EGF markedly stimulates Prss56 expression through EGFR/ERK pathway. Based on promoter analysis, we also showed that Egr2 is involved in Prss56 regulation by HB-EGF. Collectively, Prss56 expression at implantation site is modulated by HB-EGF/EGFR/ERK signaling pathway and involved in mouse decidualization.

Stomatin-like protein 2 is involved in endometrial stromal cell proliferation and differentiation during decidualization in mice and humans

The molecular mechanisms underlying endometrial stromal cell proliferation and differentiation (decidualization) are still not fully understood. This study revealed that increased Slp-2 expression is a significant factor modulating endometrial stromal cell proliferation and decidualization in both mice and humans. Our results showed a significant difference in the mRNA and protein levels between the implantation site and inter-implantation site on day 5 and day 6 of pregnancy in mice (all P < 0.05). Strong Slp-2 immunostaining was mainly localized within the decidual zone of mice through the post-implantation period. Mice with artificially induced deciduoma showed significantly higher expression of Slp-2 compared with uninduced controls (P < 0.005). Human stromal cells in the middle and late-secretory phases demonstrated significantly (all P < 0.05) upregulated SLP-2, compared with cells in the proliferative phase and early secretory phases. Further analyses of the SLP-2 gene knocked down revealed a significant (P < 0.005) repression of both the decidualization marker gene's expression (decidual/trophoblast prolactin-related protein in mice, insulin-like growth factor binding protein and prolactin in human) and the cell proliferation in in vitro-induced decidualized primary endometrial stromal cells in mice and humans.

Positive Regulation of Decidualization by l-Type Amino Acid Transporter 1 (lat1) in Pregnant Mice.

Amino acids have an important role in the pre and post implantation of placenta and embryo development. l-type amino-acid transporter 1 (lat1) is responsible for the transportation of large neutral amino acids and is mainly expressed in human fetal liver, placenta, brain, etc. This study is the first to investigate the expression of lat1 in the early pregnancy of mouse uteri and its role in the process of decidualization. Endometrial stromal cells of a mouse model were used to evaluate decidualization from Day 4–8 of pregnancy in vitro followed by lat1 knock down by small interfering RNA and by a competitive inhibitor of Leucine transport 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The effects of lat1 on decidualization in vivo were assessed by injecting BCH into the uterine horns. The mRNA and protein expressions of lat1 in the implantation sites were higher than that in the inter-implantation sites and were localized in the luminal and gland epithelium, stromal and decidual cells. Its increased expression (p < 0.05) was associated with artificial decidualization as well as activation of prl expression. Down-regulation of lat1 expression in these cells by siRNA and BCH inhibited the decidual progression in vitro. Inhibition of lat1 transportation by BCH controlled decidual progression in vivo also accompanied the down-regulation of prl, lat1 expression in the decidual area and embryo size on Day 8 of pregnancy. In conclusion, these results revealed that lat1 might play an important role in the decidual progression both in vitro and in vivo.

Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice

Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy.

MiR-125b regulates endometrial receptivity by targeting MMP26 in women undergoing IVF-ET with elevated progesterone on HCG priming day.

On the women undergoing IVF-ET with elevated progesterone on human chorionic gonadotrophin priming, the assisted reproductive technology outcome is poor. But, due to the unknown mechanism of this process, no effective method has been found to overcome this difficulty. Here, we investigated the roles of miR-125b and its target gene, MMP26, in endometrial receptivity (ER) in these women. The expression of miR-125b was significantly up-regulated in EECs in women with elevated progesterone during the window of implantation, and it showed a progesterone-dependent effect in vitro. Similarly, the expression of miR-125b was significantly up-regulated in the preimplantation period, and was down-regulated in the implantation period and the post-implantation period in mouse EECs. In addition, miR-125b showed a greater decrease at implantation sites than it did at interimplantation sites. The luciferase report assay demonstrated that MMP26 is a target gene of miR-125b. And the expression profile of MMP26 showed an inverse relationship with miR-125b in vivo and in vitro. Overexpression of miR-125b in human EECs inhibited cell migration and invasion. Gain-of-function of miR-125b induced a significant decrease in the number of implantation sites. In conclusion, these data shed new light on how miR-125b triggers ER decline through the regulation of MMP26 function.

Identification of gene expression changes in rabbit uterus during embryo implantation

Embryo implantation in the rabbit is unique in that a typical fusion type of implantation is employed, in which trophoblast cells adhere and fuse to the apical surface of uterine epithelial cells. In the present study, we analyzed global gene expression changes in the rabbit uterus during embryo implantation by using RNA-seq. We identified a total of 536 differentially expressed genes (fold change >2 and adjusted p-value <0.01), of which 266 genes were down-regulated and 270 genes were up-regulated at the implantation site compared to the inter-implantation site. Functional clustering revealed that cell adhesion is among top ranked enriched terms from both gene ontology and pathway analysis, highlighting the importance of cell adhesion during embryo implantation in rabbits. Through gene network analysis, we prioritized 9 genes using the hub gene method. Our study provides a valuable resource for in-depth understanding of the mechanism underlying embryo implantation in rabbits.

Mouse Endometrium Temporal and Spatial Expression mRNA and MicroRNA Associated With Embryo Implantation.

Embryo implantation is a dynamic physiological process involving morphological and molecular changes in the endometrium during the pre-receptivity, receptivity, and implantation phases. A comprehensive analysis of messenger RNA (mRNA) and microRNA (miRNA) profiles during implantation will likely provide new clues to elucidate the underlying mechanisms governing embryo implantation. We characterized the mRNA and miRNA transcriptomes using next generation sequencing (NGS) of the endometrium 1 day postcoitum (dpc) and 4dpc and the implantation site (IMS) and inter-implantation (IIM) site of the endometrium on 5dpc. Real-time quantitative polymerase chain reaction was performed on selected miRNAs and their predicted target mRNAs to validate their negatively correlated expression. Statistical analysis of the data based on Gene Ontology (GO) group annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the genes with significant expression at the IIM site were primarily involved in glucose, protein, and lipoprotein metabolism to provide energy for embryo implantation, while the genes identified at the IMS were involved in RNA functions to produce proteins in support of embryo development and trophoblast invasion. Extracellular matrix (ECM)-receptor interactions between cells and the ECM was the most remarkable event during implantation. The miRNA-mRNA interaction network unraveled the regulatory relationship between miRNAs and mRNAs. Hub miRNAs (mmu-miR-96 and mmu-miR-200b) were identified to target B-cell lymphoma 2 (Bcl-2), Kruppel-like factor 13 (Klf13), and Progesterone receptor (PGR), which are associated with the preparation of the receptive condition or the maintenance of early pregnancy.

Annexin A2 Acts as an Adhesion Molecule on the Endometrial Epithelium during Implantation in Mice.

To determine the function of Annexin A2 (Axna2) in mouse embryo implantation in vivo, experimental manipulation of Axna2 activities was performed in mouse endometrial tissue in vivo and in vitro. Histological examination of endometrial tissues was performed throughout the reproduction cycle and after steroid treatment. Embryo implantation was determined after blockage of the Axna2 activities by siRNA or anti-Axna2 antibody. The expression of Axna2 immunoreactivies in the endometrial luminal epithelium changed cyclically in the estrus cycle and was upregulated by estrogen. After nidatory estrogen surge, there was a concentration of Axna2 immunoreactivities at the interface between the implanting embryo and the luminal epithelium. The phenomenon was likely to be induced by the implanting embryos as no such concentration of signal was observed in the inter-implantation sites and in pseudopregnancy. Knockdown of Axna2 by siRNA reduced attachment of mouse blastocysts onto endometrial tissues in vitro. Consistently, the number of implantation sites was significantly reduced after infusion of anti-Axna2 antibody into the uterine cavity. Steroids and embryos modulate the expression of Axna2 in the endometrial epithelium. Axna2 may function as an adhesion molecule during embryo implantation in mice.

Mmu-miR-126a-3p plays a role in murine embryo implantation by regulating Itga11

miR-126a-3p has been found to be specifically up-regulated in the process of murine embryo implantation. This study aimed to further clarify the role of miR-126a-3p in embryo implantation. The expression of miR-126a-3p in implantation sites was significantly higher than that in interimplantation sites (P = 0.009). Its expression dynamics in a series of models, including pseudopregnancy, delayed implantation and artificial decidualization, suggested that the induction of miR-126a-3p is dependent on hormonal signalling and decidualization of the endometrium. Bioinformatic analysis predicted and luciferase activity assay confirmed that Itga11, a member of the integrin family, was a target gene of miR-126a-3p. miR-126a-3p bound to the 3′ untranslated region of Itga11 and regulated Itga11 by inhibiting mRNA translation and affecting mRNA stability. Transwell assay showed that miR-126a-3p promoted cell migratory and invasive capacity in vitro. Loss of function of miR-126a-3p significantly reduced the number of implantation sites in vivo (P = 0.013). Collectively, miR-126a-3p may play a major role in embryo implantation by regulating Itga11, possibly through impairing cell migratory and invasive capacity. These findings should contribute to a better understanding of the miRNA-based mechanism of embryo implantation.

Transcriptome of early embryonic invasion at implantation sites in a murine model.

Successful implantation relies on the interaction between a competent embryo and a receptive endometrium. The aim of the present study was to investigate genes differentially expressed in early invasive embryonic tissue versus decidual tissue in mice. Samples were obtained from the ectoplacental cone, the immediately surrounding deciduas and from deciduas from interimplantation sites. Microarray analysis showed that 817 genes were differentially expressed between extra-embryonic tissue and the surrounding decidua and that 360 genes were differentially expressed between the different deciduas, with a high representation of developmental processes. Genes differentially expressed in the maternal compartment included chemokines, lipoproteins, growth factors and transcription factors, whereas the embryonic invasive tissue expressed genes commonly observed in invasive tumour-like processes. These results provide information about genes involved in early embryonic invasion and the control exerted by the surrounding decidua. This information may be useful to find targets involved in pathologies associated with implantation failure and early pregnancy loss.

The Differential Expression of MicroRNAs Between Implantation Sites and Interimplantation Sites in Early Pregnancy in Mice and Their Potential Functions

Embryo implantation is a complex process that involves synchronized crosstalk between a receptive endometrium and a functional blastocyst. It can take place only during the window of implantation, a period when a series of changes in gene expression occur in the endometrium to accept the embryo. As modulators of gene expression, microRNAs (miRNAs) have been identified as regulators of embryo implantation. To better understand how miRNAs regulate implantation and the related molecular mechanisms, we compared the expression profiles of miRNAs and messenger RNAs between implantation sites (IMs) and inter-IMs in the endometrium of pregnant mice on day 5 by microarrays. The results showed that compared with inter-IMs, 30 miRNAs were upregulated and 42 miRNAs (>2-fold) were downregulated at the IMs. By combining the results of the microarray experiments, we found that 20 upregulated pathways and 14 downregulated pathways might be subject to miRNA regulation at IMs. We also found that some miRNAs and their targets may play a key role in implantation.

The PI3K/Akt signaling pathway exerts effects on the implantation of mouse embryos by regulating the expression of RhoA

The aim of this study was to investigate whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway affects the implantation of mouse embryos by regulating the expression of RhoA. The expression of PI3K, Akt, phosphorylated (p-)Akt, phosphatase and tensin homolog (PTEN) and RhoA in the uterus of mice on day 5 of pregnancy (D5) and in pseudopregnant mice was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry and western blot analysis. A functional analysis of these genes was also performed by the intrauterine injection with the PI3K inhibitor, LY294002, on day 2 of pregnancy (D2). The expression levels of PI3K, p-Akt, RhoA at the implantation site were higher than those at the inter-implantation site in the endometrium; however, opposite effects were observed for PTEN expression. The expression levels of the above genes in the pseudopregnant group and in the group injected with the PI3K/Akt inhibitor, LY294002, were markedly lower than those in the pregnant group. Functional experiments revealed that the number of implantation sites had been significantly decreased (P<0.05) following the intrauterine injection of the PI3K inhibitor, LY294002, on day 2 of gestation compared with the contralateral injection of phosphate-buffered saline (PBS). These results suggest that the PI3K/Akt signaling pathway affects embryo implantation by regulating the expression of RhoA.

Cyclic adenosine monophosphate-induced argininosuccinate synthase 1 expression is essential during mouse decidualization

L-Arginine (L-Arg), a conditional essential amino acid in adults, has been shown to enhance pregnancy outcome. Argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) are the key enzyme for L-Arginine (L-Arg) biosynthesis. Based our microarray analysis, Ass1 expression is upregulated significantly at implantation site on day 5 of pregnancy compared to that at inter-implantation site. However, the expression, regulation and function of Ass1 during early pregnancy remain unknown. Here we found that Ass1 is highly expressed in mouse decidua and uterine stromal cells undergoing decidualization, and Asl is weakly expressed in mouse decidua and uterine stromal cells undergoing decidualization. α-Methyl-DL-aspartic acid (MDLA), a specific inhibitor for Ass1, can significantly increase the rate of embryonic reabsorption. Under in vitro induced decidualization, MDLA clearly inhibits the expression of decidual/trophoblast prolactin-related protein (Dtprp), a marker for decidualization in mice. Only Ass1 expression is induced by cAMP through PKA/p-Creb signaling pathway. Results from our cell culture models further indicates that the high level of L-Arg enhances stromal proliferation, while enzymatic activity or Ass1 expression level is essential to determine the magnitude of both mouse and human decidualization. Interestingly, L-Arg at high concentration down-regulates Ass1 and Asl expression by negative feedback to maintain L-Arg homeostasis. These findings highlight that cAMP-induced Ass1 expression is important in controlling the magnitude of decidualization through regulating L-Arg level.