ADAMTS4 is a crucial proteolytic enzyme for versican cleavage in the amnion at parturition.

Hyalectan cleavage may play an important role in extracellular matrix remodeling. However, the proteolytic enzyme responsible for hyalectan degradation for fetal membrane rupture at parturition remains unknown. Here, we reveal that versican (VCAN) is the major hyalectan in the amnion, where its cleavage increases at parturition with spontaneous rupture of membrane. We further reveal that ADAMTS4 is a crucial proteolytic enzyme for VCAN cleavage in the amnion. Inflammatory factors may enhance VCAN cleavage by inducing ADAMTS4 expression and inhibiting ADAMTS4 endocytosis in amnion fibroblasts. In turn, versikine, the VCAN cleavage product, induces inflammatory factors in amnion fibroblasts, thereby forming a feedforward loop between inflammation and VCAN degradation. Mouse studies show that intra-amniotic injection of ADAMTS4 induces preterm birth along with increased VCAN degradation and proinflammatory factors abundance in the fetal membranes. Conclusively, there is enhanced VCAN cleavage by ADAMTS4 in the amnion at parturition, which can be reenforced by inflammation.

Attenuation of palmitic acid-induced lysyl oxidase overexpression in the ovary contributes to the improvement of ovulation in obesity by metformin.

STUDY QUESTION: Does palmitic acid (PA), the most common saturated free fatty acid (FFA) in individuals with obesity, contribute to anovulation through upregulation of the collagen-crosslinking enzyme lysyl oxidase (LOX) in the ovary? SUMMARY ANSWER: Increased PA in individuals with obesity can cause LOX upregulation via the activation of hypoxia-inducible factor-1α (HIF-1α), resulting in abnormal collagen deposition in the ovary and anovulation, which can be ameliorated by metformin therapy. WHAT IS KNOWN ALREADY: The underlying cause of anovulation in individuals with obesity is poorly defined, and accumulating evidence indicates that hormonal disturbance, insulin resistance, and inflammation may all play a role in the development of ovulation disorders in individuals with obesity. However, it remains to be determined whether PA plays a role in the regulation of LOX expression, thus disrupting ovarian extracellular matrix (ECM) remodelling in the ovary and resulting in impaired ovulation in individuals with obesity. STUDY DESIGN SIZE DURATION: PA concentration and LOX protein abundance and activity in follicular fluid and ovarian tissue were compared between control (n = 21) subjects, patients with obesity with ovulation (n = 22), and patients with obesity with anovulation (n = 16). The effect of PA on LOX protein expression, and the underlying mechanism, was examined in primary human granulosa cells in vitro. The improvements in obesity conditions induced by LOX inhibition combined with metformin were investigated in a high-fat diet-induced obese rat model. PARTICIPANTS/MATERIALS SETTING METHODS: The abundance of PA concentration and LOX activity was measured via a LOX activity assay and ELISA, respectively. The effect of PA on LOX protein expression was examined in the presence or absence of inhibitors of signalling molecules and siRNA-mediated knockdown of the putative transcription factor. Chromatin immunoprecipitation assays were subsequently conducted to further identify the responsible transcription factor. The role of metformin in the treatment of anovulation by LOX inhibition was investigated in a high-fat diet (HFD)-induced obese rat model. The numbers of retrieved total oocytes and metaphase II oocytes were recorded upon ovarian stimulation. Masson's trichrome staining was used to measure the total collagen content, and immunohistochemical staining and western blotting were used to measure LOX, HIF-1α, and collagen I and IV in the ovary. MAIN RESULTS AND THE ROLE OF CHANCE: Significantly increased FFA, LOX, and collagen abundance were observed in the ovaries of obese women with anovulation, compared to healthy controls or obese women with ovulation. In a HFD-induced obese rat model, metformin corrected the distortion of ovarian morphology by decreasing LOX and collagen protein abundance in the ovary and improving oestrous cyclicity and ovulation. PA increased LOX expression via the activation of HIF-1α in human granulosa cells, which was attenuated by metformin. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Several other saturated and polyunsaturated FFAs, such as stearic acid and arachidonic acid, are also increased in the blood of individuals with obesity, and increased levels of other FFAs may also contribute to the development of anovulation in individuals with obesity, which needs to be further verified in the future. WIDER IMPLICATIONS OF THE FINDINGS: Elevated PA in individuals with obesity can cause LOX dysregulation via activation of HIF-1α, resulting in abnormal collagen deposition in the ovary and anovulation. This dysregulation can be ameliorated by metformin therapy through its local effect on ECM remodelling in the ovary, which is independent of its systemic effect on insulin sensitivity and chronic inflammation. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the National Natural Science Foundation of China (grant numbers 82101730, 82130046, and 31900598) and Innovative Research Team of High-level local Universities in Shanghai (SHSMU-ZLCX20210201). All the authors declare no conflicts of interest in relation to this work.

The Role of DNMT1 and C/EBPα in the Regulation of CYP11A1 Expression During Syncytialization of Human Placental Trophoblasts.

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization.

Paradoxical Induction of ALOX15/15B by Cortisol in Human Amnion Fibroblasts: Implications for Inflammatory Responses of the Fetal Membranes at Parturition.

Inflammation of the fetal membranes is an indispensable event of parturition, with increasing prostaglandin E2 (PGE2) synthesis as one of the ultimate products that prime labor onset. In addition to PGE2, the fetal membranes also boast a large capacity for cortisol regeneration. It is intriguing how increased PGE2 synthesis is achieved in the presence of increasing amounts of classical anti-inflammatory glucocorticoids in the fetal membranes at parturition. 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) synthesized by lipoxygenase 15/15B (ALOX15/15B) has been shown to enhance inflammation-induced PGE2 synthesis in amnion fibroblasts. Here, we examined whether glucocorticoids could induce ALOX15/15B expression and 15(S)-HETE production to promote PGE2 synthesis in amnion fibroblasts at parturition. We found that cortisol and 15(S)-HETE abundance increased parallelly in the amnion at parturition. Cortisol induced ALOX15/15B expression and 15(S)-HETE production paradoxically in amnion fibroblasts. Mechanism study revealed that this paradoxical induction was mediated by p300-mediated histone acetylation and interaction of glucocorticoid receptor with transcription factors CREB and STAT3. Conclusively, cortisol regenerated in the fetal membranes can paradoxically induce ALOX15/15B expression and 15(S)-HETE production in human amnion fibroblasts, which may further assist in the induction of PGE2 synthesis in the inflammatory responses of the fetal membranes for parturition.

IL-33/ST2 axis of human amnion fibroblasts participates in inflammatory reactions at parturition.

BACKGROUND: Inflammation of the fetal membranes is an indispensable event of labor onset at both term and preterm birth. Interleukin-33 (IL-33) is known to participate in inflammation via ST2 (suppression of tumorigenicity 2) receptor as an inflammatory cytokine. However, it remains unknown whether IL-33/ST2 axis exists in human fetal membranes to promote inflammatory reactions in parturition. METHODS: The presence of IL-33 and ST2 and their changes at parturition were examined with transcriptomic sequencing, quantitative real-time polymerase chain reaction, Western blotting or immunohistochemistry in human amnion obtained from term and preterm birth with or without labor. Cultured primary human amnion fibroblasts were utilized to investigate the regulation and the role of IL-33/ST2 axis in the inflammation reactions. A mouse model was used to further study the role of IL-33 in parturition. RESULTS: Although IL-33 and ST2 expression were detected in both epithelial and fibroblast cells of human amnion, they are more abundant in amnion fibroblasts. Their abundance increased significantly in the amnion at both term and preterm birth with labor. Lipopolysaccharide, serum amyloid A1 and IL-1ß, the inflammatory mediators pertinent to labor onset, could all induce IL-33 expression through NF-κB activation in human amnion fibroblasts. In turn, via ST2 receptor, IL-33 induced the production of IL-1ß, IL-6 and PGE2 in human amnion fibroblasts via the MAPKs-NF-κB pathway. Moreover, IL-33 administration induced preterm birth in mice. CONCLUSION: IL-33/ST2 axis is present in human amnion fibroblasts, which is activated in both term and preterm labor. Activation of this axis leads to increased production of inflammatory factors pertinent to parturition, and results in preterm birth. Targeting the IL-33/ST2 axis may have potential value in the treatment of preterm birth.

Amnion-derived serum amyloid A1 participates in sterile inflammation of fetal membranes at parturition.

OBJECTIVES: Sterile inflammation of fetal membranes is an indispensable event of normal parturition. However, triggers of sterile inflammation are not fully resolved. Serum amyloid A1 (SAA1) is an acute phase protein produced primarily by the liver. Fetal membranes can also synthesize SAA1 but its functions are not well defined. Given the role of SAA1 in the acute phase response to inflammation, we postulated that SAA1 synthesized in the fetal membranes may be a trigger of local inflammation at parturition. METHODS: The changes of SAA1 abundance in parturition were studied in the amnion of human fetal membranes. The role of SAA1 in chemokine expression and leukocyte chemotaxis was examined in cultured human amnion tissue explants as well as primary human amnion fibroblasts. The effects of SAA1 on monocytes, macrophages and dendritic cells were investigated in cells derived from a human leukemia monocytic cell line (THP-1). RESULTS: SAA1 synthesis increased significantly in human amnion at parturition. SAA1 evoked multiple chemotaxis pathways in human amnion fibroblasts along with upregulation of a series of chemokines via both toll-like receptor 4 (TLR4) and formyl peptide receptor 2 (FPR2). Moreover, SAA1-conditioned medium of cultured amnion fibroblasts was capable of chemoattracting virtually all types of mononuclear leukocytes, particularly monocytes and dendritic cells, which reconciled with the chemotactic activity of conditioned medium of cultured amnion tissue explants collected from spontaneous labor. Furthermore, SAA1 could induce the expression of genes associated with inflammation and extracellular matrix remodeling in monocytes, macrophages and dendritic cells derived from THP-1. CONCLUSIONS: SAA1 is a trigger of sterile inflammation of the fetal membranes at parturition.

ER-phagy Is Involved in the Degradation of Collagen I by IL-1ß in Human Amnion in Parturition.

The process of parturition is associated with inflammation within the uterine tissues, and IL-1ß is a key proinflammatory cytokine involved. Autophagy is emerging as an important pathway to remove redundant cellular components. However, it is not known whether IL-1ß employs the autophagy pathway to degrade collagen, thereby participating in membrane rupture at parturition. In this study, we investigated this issue in human amnion. Results showed that IL-1ß levels were significantly increased in human amnion obtained from deliveries with spontaneous labor and membrane rupture, which was accompanied by decreased abundance of COL1A1 and COL1A2 protein but not their mRNA, the two components of collagen I. Consistently, IL-1ß treatment of cultured primary human amnion fibroblasts reduced COL1A1 and COL1A2 protein but not their mRNA abundance along with increased abundance of autophagy activation markers, including the microtubule-associated protein L chain 3ß II/I ratio and autophagy-related 7 (ATG7) in the cells. The reduction in COL1A1 and COL1A2 protein abundance induced by IL-1ß could be blocked by the lysosome inhibitor chloroquine or small interfering RNA-mediated knockdown of ATG7 or ER-phagy receptor FAM134C, suggesting that FAM134C-mediated ER-phagy was involved in IL-1ß-induced reduction in COL1A1 and COL1A2 protein in amnion fibroblasts. Consistently, levels of L chain 3ß II/I ratio, ATG7, and FAM134C were significantly increased in human amnion obtained from deliveries with spontaneous labor and membrane rupture. Conclusively, increased IL-1ß abundance in human amnion may stimulate ER-phagy-mediated COL1A1 and COL1A2 protein degradation in amnion fibroblasts, thereby participating in membrane rupture at parturition.

Identification of a Feed-Forward Loop Between 15(S)-HETE and PGE2 in Human Amnion at Parturition.

Human parturition is associated with massive arachidonic acid (AA) mobilization in the amnion, indicating that large amounts of AA-derived eicosanoids are required for parturition. Prostaglandin E2 (PGE2) synthesized from the cyclooxygenase (COX) pathway is the best characterized AA-derived eicosanoid in the amnion which plays a pivotal role in parturition. The existence of any other pivotal AA-derived eicosanoids involved in parturition remains elusive. Here, we screened such eicosanoids in human amnion tissue with AA-targeted metabolomics and studied their role and synthesis in parturition by using human amnion fibroblasts and a mouse model. We found that lipoxygenase (ALOX) pathway-derived 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) and its synthetic enzymes ALOX15 and ALOX15B were significantly increased in human amnion at parturition. Although 15(S)-HETE is ineffective on its own, it potently potentiated the activation of NF-κB by inflammatory mediators including lipopolysaccharide, interleukin-1ß, and serum amyloid A1, resulting in the amplification of COX-2 expression and PGE2 production in amnion fibroblasts. In turn, we determined that PGE2 induced ALOX15/15B expression and 15(S)-HETE production through its EP2 receptor-coupled PKA pathway, thereby forming a feed-forward loop between 15(S)-HETE and PGE2 production in the amnion at parturition. Our studies in pregnant mice showed that 15(S)-HETE injection induced preterm birth with increased COX-2 and PGE2 abundance in the fetal membranes and placenta. Conclusively, 15(S)-HETE is identified as another crucial parturition-pertinent AA-derived eicosanoid in the amnion, which may form a feed-forward loop with PGE2 in parturition. Interruption of this feed-forward loop may be of therapeutic value for the treatment of preterm birth.

ER-phagy Is Involved in the Degradation of Collagen I by IL-1ß in Human Amnion in Parturition.

The process of parturition is associated with inflammation within the uterine tissues, and IL-1ß is a key proinflammatory cytokine involved. Autophagy is emerging as an important pathway to remove redundant cellular components. However, it is not known whether IL-1ß employs the autophagy pathway to degrade collagen, thereby participating in membrane rupture at parturition. In this study, we investigated this issue in human amnion. Results showed that IL-1ß levels were significantly increased in human amnion obtained from deliveries with spontaneous labor and membrane rupture, which was accompanied by decreased abundance of COL1A1 and COL1A2 protein but not their mRNA, the two components of collagen I. Consistently, IL-1ß treatment of cultured primary human amnion fibroblasts reduced COL1A1 and COL1A2 protein but not their mRNA abundance along with increased abundance of autophagy activation markers, including the microtubule-associated protein L chain 3ß II/I ratio and autophagy-related 7 (ATG7) in the cells. The reduction in COL1A1 and COL1A2 protein abundance induced by IL-1ß could be blocked by the lysosome inhibitor chloroquine or small interfering RNA-mediated knockdown of ATG7 or ER-phagy receptor FAM134C, suggesting that FAM134C-mediated ER-phagy was involved in IL-1ß-induced reduction in COL1A1 and COL1A2 protein in amnion fibroblasts. Consistently, levels of L chain 3ß II/I ratio, ATG7, and FAM134C were significantly increased in human amnion obtained from deliveries with spontaneous labor and membrane rupture. Conclusively, increased IL-1ß abundance in human amnion may stimulate ER-phagy-mediated COL1A1 and COL1A2 protein degradation in amnion fibroblasts, thereby participating in membrane rupture at parturition.

Cortisol Stimulates Local Progesterone Withdrawal Through Induction of AKR1C1 in Human Amnion Fibroblasts at Parturition.

Fetal membrane activation is seen as being one of the crucial triggering components of human parturition. Increased prostaglandin E2 (PGE2) production, a common mediator of labor onset in virtually all species, is recognized as one of the landmark events of membrane activation. Fetal membranes are also equipped with a high capacity of cortisol regeneration by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), and the cortisol regenerated potently induces PGE2 synthesis, an effect normally suppressed by progesterone during gestation. There is no precipitous decline of progesterone synthesis in human parturition. It is intriguing how this suppression is lifted in parturition. Here, we investigated this issue by using human amnion tissue and primary amnion fibroblasts which synthesize the most PGE2 in the fetal membranes. Results showed that the expression of 11ß-HSD1 and aldo-keto reductase family 1 member C1 (AKR1C1), a progesterone-inactivating enzyme, increased in parallel in human amnion tissue with gestational age toward the end of gestation and at parturition. Cortisol induced AKR1C1 expression via the transcription factor CCAAT enhancer binding protein δ (C/EBPδ) in amnion fibroblasts. Inhibition of AKR1C1 not only blocked progesterone catabolism induced by cortisol, but also enhanced the suppression of cortisol-induced cyclooxygenase-2 (COX-2) expression by progesterone in amnion fibroblasts. In conclusion, our results indicate that cortisol regenerated in the fetal membranes triggers local progesterone withdrawal through enhancement of AKR1C1-mediated progesterone catabolism in amnion fibroblasts, so that the suppression of progesterone on the induction of COX-2 expression and PGE2 synthesis by cortisol can be lifted for parturition.

Serum amyloid A, a host-derived DAMP in pregnancy?

Serum amyloid A (SAA) is one of the acute phase proteins released primarily from the liver in response to infection, inflammation and trauma. Emerging evidence indicates that SAA may function as a host-derived damage-associated molecular pattern (DAMP) protein to sense danger signals in pregnancy. The plasma SAA levels in maternal circulation are significantly increased in normal parturition, particularly in postpartum, as well as in gestational disorders such as premature preterm rupture of membranes, pre-eclampsia, gestational diabetes, and recurrent spontaneous abortion. It is likely that SAA acts as a non-specific DAMP molecule in response to inflammation and trauma experienced under these conditions. Notably, SAA can also be synthesized locally in virtually all gestational tissues. Within these gestational tissues, under the induction by bacterial products, pro-inflammatory cytokines and stress hormone glucocorticoids, SAA may exert tissue-specific effects as a toll-like receptor 4 (TLR4)-sensed DAMP molecule. SAA may promote parturition through stimulation of inflammatory reactions via induction of pro-inflammatory cytokines, chemokines, adhesion molecules and prostaglandins in the uterus, fetal membranes and placenta. In the fetal membranes, SAA may also facilitate membrane rupture through induction of matrix metalloproteases (MMPs)- and autophagy-mediated collagen breakdown and attenuation of lysyl oxidase-mediated collagen cross-linking. SAA synthesized in extravillous trophoblasts may promote their invasiveness into the endometrium in placentation. Here, we summarized the current understanding of SAA in pregnancy with an aim to stimulate in-depth investigation of SAA in pregnancy, which may help better understand how inflammation is initiated in gestational tissues in both normal and abnormal pregnancies.

Single cell transcriptomic analysis of human amnion identifies cell-specific signatures associated with membrane rupture and parturition.

BACKGROUND: The human amnion is an intrauterine tissue which is involved in the initiation of parturition. In-depth understanding of gene expression signatures of individual cell types in the amnion with respect to membrane rupture at parturition may help identify crucial initiators of parturition for the development of specific strategies to prevent preterm birth, a leading cause of perinatal mortality. RESULTS: Six major cell types were revealed in human amnion including epithelial cells, fibroblasts and immunocytes as well as three other cell types expressing dual cell markers including epithelial/fibroblast, immune/epithelial and immune/fibroblast markers. The existence of cell types expressing these dual cell markers indicates the presence of epithelial-mesenchymal (EMT), epithelial-immune (EIT) and mesenchymal-immune (MIT) transitions in amnion at parturition. We found that the rupture zone of amnion exhibited some specific increases in subcluster proportions of immune and EMT cells related to extracellular matrix remodeling and inflammation in labor. The non-rupture zone exhibited some common changes in subcluster compositions of epithelial and fibroblast cells with the rupture zone in labor, particularly those related to oxidative stress and apoptosis in epithelial cells and zinc ion transport in fibroblasts. Moreover, we identified that C-C motif chemokine ligand 20 (CCL20) was among the top up-regulated genes in amnion epithelial cells, fibroblasts and immunocytes in the rupture zone at parturition. Studies in pregnant mice showed that administration of CCL20 induced immunocytes infiltration to tissues at the maternal-fetal interface and led to preterm birth. CONCLUSIONS: Apart from the conventional epithelial, fibroblast and immunocytes, human amnion cells may undergo EMT, EIT and FIT in preparation for parturition. Intense inflammation and ECM remodeling are present in the rupture zone, while enhanced apoptosis and oxidative stress in epithelial cells and zinc ion transport in fibroblasts are present in amnion regardless of the rupture zones at parturition. CCL20 derived from the major cell types of the amnion participates in labor onset.

The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth.

Background: Bradykinin (BK) and its biologically active metabolite des-Arg9 bradykinin (DABK) play a pivotal role in inflammation. Since chorioamnionitis is the leading cause of preterm birth and prostaglandin E2 (PGE2) derived from the amnion is key to labor initiation, we investigated if bradykinin peptides are part of the regulatory network of PGE2 synthesis in human amnion at parturition. Methods: Human amnion tissue was obtained from term and preterm birth for the study of the changes of the bradykinin system at parturition. Cultured primary human amnion fibroblasts, the major source of PGE2, were used to study the effects of bradykinin peptides on PTGS2 expression and PGE2 production as well as the effects of infection mediators on bradykinin receptors. Results: Bradykinin peptides and their receptors BDKRB1 and BDKRB2 were present in human amnion, and their abundance increased in term and preterm labor. However, transcripts of the genes encoding the bradykinin precursor and its proteolytic cleavage enzymes were hardly detectable in human amnion despite the increased abundance of bradykinin peptides in term and preterm labor, suggesting that there is an alternative source of bradykinin peptides for human amnion and their actions are enhanced in human amnion at parturition. In-vitro studies in cultured human amnion fibroblasts showed that both BK and DABK increased the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the rate-limiting enzyme in prostaglandin synthesis, and subsequent PGE2 production. These effects of BK and DABK were mediated through BDKRB2 and BDKRB1 receptors, respectively, with subsequent activation of the p38 and ERK1/2 pathways. Moreover, lipopolysaccharide (LPS) and serum amyloid A1 (SAA1), the important mediators of infectious inflammation, induced the expression of both BDKRB1 and BDKRB2 through toll-like receptor 4 (TLR4). Induction of BDKRB1 and BDKRB2 expression by LPS and SAA1 enhanced BK- or DABK-induced PTGS2 expression and PGE2 production in human amnion fibroblasts. Conclusions: This study demonstrated for the first time that the human amnion is a target tissue of bradykinin peptides and the bradykinin system may be part of the regulatory network of PTGS2 expression and PGE2 production in human amnion fibroblasts at both term and preterm birth, which may be enhanced by infection.

Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2)-mediated histone 3 lysine 27 trimethylation (H3K27me3) is a transcription silencing mark, which is indispensable for cell lineage specification at the early blastocyst stage. This epigenetic repression is maintained in placental cytotrophoblasts but is lifted when cytotrophoblasts differentiate into syncytiotrophoblasts. However, the physiological impact of this lift remains elusive. Here, we investigated whether lifting EZH2-mediated H3K27me3 during syncytialization upregulates the expression of a short secretory isoform of a disintegrin and metalloprotease 12 (ADAM12-S), a well-recognized placenta-derived protease that cleaves insulin-like growth factor binding protein 3 to increase insulin-like growth factor (IGF) bioavailability for the stimulation of fetoplacental growth. The transcription factor and the upstream signal involved were also explored. METHODS: Human placenta tissue and cultured primary human placental cytotrophoblasts were utilized to investigate the role of EZH2-mediated H3K27me3 in ADAM12-S expression and the associated transcription factor and upstream signal during syncytialization. A mouse model was used to examine whether inhibition of EZH2-mediated H3K27me3 regulates placental ADAM12-S expression and fetoplacental growth. RESULTS: EZH2 and ADAM12 are distributed primarily in villous cytotrophoblasts and syncytiotrophoblasts, respectively. Increased ADAM12-S expression, decreased EZH2 expression, and decreased EZH2/H3K27me3 enrichment at the ADAM12 promoter were observed during syncytialization. Knock-down of EZH2 further increased ADAM12-S expression in trophoblasts. Syncytialization was also accompanied by increased STAT5B expression and phosphorylation as well as its enrichment at the ADAM12 promoter. Knock-down of STAT5B attenuated ADAM12-S expression during syncytialization. Epidermal growth factor (EGF) was capable of inducing ADAM12-S expression via stimulation of STAT5B expression and phosphorylation during syncytialization. Mouse studies revealed that administration of an EZH2 inhibitor significantly increased ADAM12-S levels in maternal blood and fetoplacental weights along with decreased H3K27me3 abundance and increased ADAM12-S expression in the placenta. CONCLUSIONS: Lifting EZH2-mediated H3K27me3 increases ADAM12-S expression during syncytialization with the participation of EGF-activated STAT5B, which may lead to elevation of ADAM12-S level in maternal blood resulting in increased IGF bioavailability for the stimulation of fetoplacental growth in pregnancy. Our studies suggest that the role of EZH2-mediated H3K27me3 may switch from cell lineage specification at the early blastocyst stage to regulation of fetoplacental growth in later gestation.

C/EBPδ drives key endocrine signals in the human amnion at parturition.

Amnion-derived prostaglandin E2 (PGE2) and cortisol are key to labor onset. Identification of a common transcription factor driving the expression of both cyclooxygenase-2 (COX-2) and 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), the key enzymes in their production, may hold the key to the treatment of pre-term labor. Here, we have found that the CCAAT enhancer binding protein δ (C/EBPδ) is such a transcription factor which underlies the feed-forward induction of COX-2 and 11ß-HSD1 expression by their own products PGE2 and cortisol in human amnion fibroblasts so that their production would be ensured in the amnion for the onset of labor. Moreover, the abundance of C/EBPδ in the amnion increases along with COX-2 and 11ß-HSD1 at term and further increases at parturition. Knockout of C/EBPδ in mice delays the onset of labor further supporting the concept. In conclusion, C/EBPδ pathway may be speculated to serve as a potential pharmaceutical target in the amnion for treatment of pre-term labor.

PGE2 vs PGF2α in human parturition.

Prostaglandin E2 (PGE2) and F2α (PGF2α) are the two most prominent prostanoids in parturition. They are involved in cervical ripening, membrane rupture, myometrial contraction and inflammation in gestational tissues. Because multiple receptor subtypes for PGE2 and PGF2α exist, coupled with diverse signaling pathways, the effects of PGE2 and PGF2α depend largely on the spatial and temporal expression of these receptors in intrauterine tissues. It appears that PGE2 and PGF2α play different roles in parturition. PGE2 is probably more important for labor onset, while PGF2α may play a more important role in labor accomplishment, which may be attributed to the differential effects of PGE2 and PGF2α in gestational tissues. PGE2 is more powerful than PGF2α in the induction of cervical ripening. In terms of myometrial contraction, PGE2 produces a biphasic effect with an initial contraction and a following relaxation, while PGF2α consistently stimulates myometrial contraction. In the fetal membranes, both PGE2 and PGF2α appear to be involved in the process of membrane rupture. In addition, PGE2 and PGF2α may also participate in the inflammatory process of intrauterine tissues at parturition by stimulating not only neutrophil influx and cytokine production but also cyclooxygenase-2 expression thereby intensifying their own production. This review summarizes the differential roles of PGE2 and PGF2α in parturition with respect to their production and expression of receptor subtypes in gestational tissues. Dissecting the specific mechanisms underlying the effects of PGE2 and PGF2α in parturition may assist in developing specific therapeutic targets for preterm and post-term birth.

lnc-MAP3K13-7:1 Inhibits Ovarian GC Proliferation in PCOS via DNMT1 Downregulation-Mediated CDKN1A Promoter Hypomethylation.

Polycystic ovary syndrome (PCOS) is an endocrine-related disease and global cause of infertility that is associated with abnormal folliculogenesis. Inhibited granulosa cell (GC) proliferation is recognized as a key factor that underlies aberrant follicle maturation. Many epigenetic landscape modifications have been characterized in PCOS patients. However, the epigenetic regulation pathways in follicular dysplasia are not completely understood. In this study, we reported a novel mechanism of DNA hypomethylation induced by long non-coding RNAs (lncRNAs) and its function in cell cycle progression. We observed that lnc-MAP3K13-7:1 was highly expressed in GCs from patients with PCOS, with concomitant global DNA hypomethylation, decreased DNA methyltransferase 1 (DNMT1) expression, and increased cyclin-dependent kinase inhibitor 1A (CDKN1A, p21) expression. In KGN cells, lnc-MAP3K13-7:1 overexpression resulted in cell cycle arrest in the G0/G1 phase, as well as the molecular inhibition and genetic silencing of DNMT1. Mechanistically, lnc-MAP3K13-7:1 inhibited DNMT1 expression by acting as a protein-binding scaffold and inducing ubiquitin-mediated DNMT1 protein degradation. Moreover, DNMT1-dependent CDKN1A promoter hypomethylation increased CDKN1A transcription, resulting in attenuated GC growth. Our work uncovered a novel and essential mechanism through which lnc-MAP3K13-7:1-dependent DNMT1 inhibition regulates CDKN1A/p21 expression and inhibits GC proliferation.

De novo Synthesis of SAA1 in the Placenta Participates in Parturition.

Serum amyloid A1 (SAA1) is an acute phase protein produced mainly by the liver to participate in immunomodulation in both sterile and non-sterile inflammation. However, non-hepatic tissues can also synthesize SAA1. It remains to be determined whether SAA1 synthesized locally in the placenta participates in parturition via eliciting inflammatory reactions. In this study, we investigated this issue by using human placenta and a mouse model. We found that SAA1 mRNA and protein were present in human placental villous trophoblasts, which was increased upon syncytialization as well as treatments with lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α), and cortisol. Moreover, significant increases in SAA1 abundance were observed in the placental tissue or in the maternal blood in spontaneous deliveries without infection at term and in preterm birth with histological chorioamnionitis. Serum amyloid A1 treatment significantly increased parturition-pertinent inflammatory gene expression including interleukin-1ß (IL-1ß), IL-8, TNF-α, and cyclooxygenase-2 (COX-2), along with increased PGF2α production in syncytiotrophoblasts. Mouse study showed that SAA1 was present in the placental junctional zone and yolk sac membrane, which was increased following intraperitoneal administration of LPS. Intraperitoneal injection of SAA1 not only induced preterm birth but also increased the abundance of IL-1ß, TNF-α, and COX-2 in the mouse placenta. Conclusively, SAA1 can be synthesized in the human placenta, which is increased upon trophoblast syncytialization. Parturition is accompanied with increased SAA1 abundance in the placenta. Serum amyloid A1 may participate in parturition in the presence and absence of infection by inducing the expression of inflammatory genes in the placenta.

Cortisol Regeneration in the Fetal Membranes, A Coincidental or Requisite Event in Human Parturition?

The fetal membranes are equipped with high capacity of cortisol regeneration through the reductase activity of 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1). The expression of 11ß-HSD1 in the fetal membranes is under the feedforward induction by cortisol, which is potentiated by proinflammatory cytokines. As a result, the abundance of 11ß-HSD1 increases with gestational age and furthermore at parturition with an escalation of cortisol concentration in the fetal membranes. Accumulated cortisol takes parts in a number of crucial events pertinent to the onset of labor in the fetal membranes, including extracellular matrix (ECM) remodeling and stimulation of prostaglandin output. Cortisol remodels the ECM through multiple approaches including induction of collagen I, III, and IV degradation, as well as inhibition of their cross-linking. These effects of cortisol are executed through activation of the autophagy, proteasome, and matrix metalloprotease 7 pathways, as well as inhibition of the expression of cross-linking enzyme lysyl oxidase in mesenchymal cells of the membranes. With regard to prostaglandin output, cortisol not only increases prostaglandin E2 and F2α syntheses through induction of their synthesizing enzymes such as cytosolic phospholipase A2, cyclooxygenase 2, and carbonyl reductase 1 in the amnion, but also decreases their degradation through inhibition of their metabolizing enzyme 15-hydroxyprostaglandin dehydrogenase in the chorion. Taking all together, data accumulated so far denote that the feedforward cortisol regeneration by 11ß-HSD1 in the fetal membranes is a requisite event in the onset of parturition, and the effects of cortisol on prostaglandin synthesis and ECM remodeling may be enhanced by proinflammatory cytokines in chorioamnionitis.

The Importance of a Conjoint Analysis of Tumor-Associated Macrophages and Immune Checkpoints in Pancreatic Cancer.

OBJECTIVES: Tumor-associated macrophages are dominant players in establishing the inmmunosuppressive microenvironment in pancreatic ductal adenocarcinoma (PDAC). Immune checkpoint inhibitor monotherapy has achieved limited clinical effectiveness. To date, the interaction of macrophages and checkpoint regulators and their correlation with clinicopathologic characteristics in PDAC have been largely unavailable. METHODS: Macrophages and immune checkpoint expression were assessed by immunohistochemistry from 80 PDAC samples. Clinicopathologic features and the prognostic value of each marker were evaluated. In vitro changes in the expression of immune markers in cocultured macrophages and PDAC cells were detected by Western blot and immunosorbance assays. RESULTS: The macrophages marker CD163 and the checkpoint marker programmed death-ligand 1 (PD-L1) remained as the independent prognostic factors for overall survival (hazard ratio, 2.543; P = 0.017 and hazard ratio, 2.389; P = 0.021). Furthermore, integrated analysis of CD163 and PD-L1 served as more optimal indicators of survival (P = 0.000). In vitro coculture of macrophages and PDAC cells significantly increased the expression of CD163 and PD-L1, compared with monocultured counterpart (P < 0.05). CONCLUSIONS: Combined analysis of CD163 and PD-L1 was enhanced indicators of survival in PDAC patients. The interaction of macrophages and immune checkpoints implied the value of the combination therapy.