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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. [ABSTRACT FROM AUTHOR]

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 INTEREST(S) 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. [ABSTRACT FROM AUTHOR]

Problem: Rupture of fetal membranes is a crucial event at parturition, which is preceded by extensive extracellular matrix (ECM) remodeling. Our recent studies have demonstrated that the human fetal membranes are capable of de novo synthesis of serum amyloid A1 (SAA1), an acute phase protein, and the abundance of SAA1 in the amnion was increased at parturition. However, the exact role of SAA1 in human parturition remains to be established. Method of study: The effects of SAA1 on the abundance of collagenases and lysyl oxidase, the enzyme that cross‐links collagens, were investigated in culture primary human amnion fibroblasts and tissue explants with an aim to examine the involvement of SAA1 in the ECM remodeling in the amnion. Results: Serum amyloid A1 (SAA1) time‐ and dose‐dependently increased the abundance of collagenases MMP‐1, MMP‐8, and MMP‐13, while decreased the abundance of lysyl oxidase‐like 1 (LOXL1). These effects of SAA1 were attenuated by siRNA‐mediated knockdown of the Toll‐like receptor (TLR) 4 and its antagonist CLI‐095, but not by siRNA‐mediated knockdown of TLR2. Furthermore, the inhibitors for NF‐κB (JSH‐23) and mitogen‐activated protein kinases (MAPKs) p38 (SB203580) and JNK (SP600125) could also attenuate the effects of SAA1, while the inhibitor for MAPK ERK1/2 (PD 98059) could block the effects of SAA1 only on MMP‐1, MMP‐8, and LOXL1 but not on MMP‐13. Conclusion: These data highlight a possible role for SAA1 in ECM remodeling preceding membrane rupture by regulating the expression of collagenases MMP‐1, MMP‐8, MMP‐13, and LOXL1 through TLR4‐mediated activation of the NF‐κB and MAPK pathways in amnion fibroblasts. [ABSTRACT FROM AUTHOR]

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-1β, 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-1β, 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. [ABSTRACT FROM AUTHOR]

Aim: Azithromycin (AZM) is widely used to treat mycoplasma infection in pregnancy. However, there is no adequate evaluation of its side effect on the placenta. In this study, using human placental syncytiotrophoblasts and a mouse model, we investigated whether AZM use in pregnancy might adversely affect placental function and pregnancy outcome. Results: Transcriptomic analysis of AZM-treated human placental syncytiotrophoblasts showed increased expression of endoplasmic reticulum (ER) stress-related genes and decreased expression of genes for hormone production and growth factor processing. Verification studies showed that AZM increased the abundance of ER stress mediators (phosphorylated eIF2α, activating transcription factor 4 [ATF4], and C/EBP Homologous Protein [CHOP]) and decreased the abundance of enzymes involved in progesterone and estradiol synthesis ( STS , CYP11A1 , and CYP19A1 ) and insulin-like growth factor binding protein (IGFBP) cleavage ( PAPPA and ADAM12 ) in human placental syncytiotrophoblasts. Inhibition of ER stress blocked AZM-induced decreases in the expression of CYP19A1, CYP11A1, PAPPA, and ADAM12, suggesting that the inhibition of AZM on those genes' expression was secondary to AZM-induced ER stress. Further mechanism study showed that increased ATF4 in ER stress might repressively interact with C/EBPα to suppress the expression of those genes, including CEBPA itself. Mouse studies showed that AZM administration decreased fetal weights along with increased ER stress mediators and decreased levels of insulin-like growth factor, estrogen, and progesterone in the maternal blood, which could be alleviated by inhibition of ER stress. Innovation and Conclusion: These findings first support the fact that AZM, often used during pregnancy, may affect fetal growth by inhibiting crucial enzymes for estrogen and progesterone synthesis and disrupting crucial proteases for IGFBP cleavage via inducing ER stress in placental syncytiotrophoblasts.

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)