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Endometriosis is a chronic inflammatory, estrogenic disorder caused by endometrial tissue growth places other than uterine lumen, resulting in infertility and severe pelvic pain. Thymol, an extract of Thymus vulgaris, processes diverse biological properties, including anti-inflammatory, local anesthetic, decongestant, and antiseptic effects. However, the efficacy of thymol in treating endometriosis has still not been explored. Herein, this research aimed to investigate the role of thymol in the treatment of endometriosis using a murine model and Ishikawa cells. Thirty C57BL/6 mice were administered 17β-E2 (100 ng/mouse) subcutaneously for three consecutive days to induce synchronous estrus. On the last day of injection, the mice underwent surgical induction of endometriosis. After that, the mice were divided into three groups, i.e., Control (CTRL), Thymol 30 mg/kg and Thymol 60 mg/kg, receiving oral administration of either saline or thymol (30 mg/kg/d or 60 mg/kg/d, as 0.1 mL/kg/d, respectively) for a three-week duration. Each group consisted of ten mice and was evenly divided into estrus and diestrus according to the vaginal cytology on the last day of treatment. Thymol significantly (p < 0.05) reduced the weight and volume of ectopic tissue, hindered cell proliferation, and stimulated apoptosis compared to the CTRL group. Additionally, in the thymol-treated group, the levels of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, as well as the numbers of neutrophils and macrophages, were significantly (p < 0.05) decreased. Moreover, a novel role of thymol in rebalancing estrogen and progesterone (E2-P4) signaling was explored, and it was distributed in the ectopic endometrium. Next, the role of thymol on Ishikawa cells was determined. The results demonstrated that thymol significantly (p < 0.05) suppressed the E2-induced proliferation of Ishikawa cells. Furthermore, molecular docking analyses suggested that thymol potentially binds to ESR1-like estrogens, indicating its antagonistic activity against estrogens. The estrogen receptor 1 (ESR1) and its target gene expression exhibited significant (p < 0.05) downregulation, while progesterone receptor (PGR) and target genes were markedly (p < 0.05) upregulated following thymol treatment in the ectopic endometrium. Most importantly, our data revealed the minimal impact of thymol treatment on the eutopic endometrium and its crucial role in supporting pregnancy, thus indicating the safety of thymol in treating endometriosis. Overall, our study suggests that thymol holds promising therapeutic implications for endometriosis by virtue of its anti-inflammatory properties and ability to antagonize estrogen activity. [ABSTRACT FROM AUTHOR]

Fibrosis is a pathological process characterized by abnormal activation of fibroblasts with increased synthesis of extracellular matrix components, including collagens. It may lead to loss of proper tissue architecture and organ function in clinical diseases such as systemic sclerosis and liver fibrosis. Excess accumulation of collagens is considered the primary indicator of fibrosis. Notch signaling has been reported to be involved in the fibrosis of many different organs, including the liver. Our previous study showed that the uterine‐specific over‐activation of canonical Notch1 signaling in the mouse uterus (Pgrcre/+Rosa26N1ICD/+, OEx) results in complete infertility as a consequence of multiple developmental and physiological defects, together with increased collagen accumulation evidenced by Masson's staining. In this study, we further detected expressions of all 44 collagen genes in these Notch1 gain‐of‐function transgenic mice and found that 18 collagens have been largely affected. In another aspect, using an intrauterine adhesion model (IUA), we mimicked fibrosis in the mouse uterine. The results suggested that Notch receptors were upregulated only 3 days after induction, and most of the fibril‐forming collagen began to upregulate 6 days after the surgery. Furthermore, when induced IUA in the N1ICD‐OEx mice, the expression of collagens and fibrosis levels were significantly enhanced. At last, as a Notch signaling inhibitor, the γ‐secretase inhibitor N‐[N‐(3,5‐difl uorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) pretreatment could alleviate the expression of collagens and the symptoms of fibrosis. These results demonstrate that Notch signaling may play a role in upregulating collagens expression in endometrial fibrosis and might be a potential target of fibrosis therapy in the endometrium. [ABSTRACT FROM AUTHOR]

Abstract Background Sec63 is a key component of the protein translocation machinery in the mammalian endoplasmic reticulum (ER), and involved in the post-translation processing of secretory proteins. The aim of this study was to determine the expression pattern of SEC63 gene in mouse uterus during the early pregnancy. Methods Real-time quantitative PCR and Western blot analyses were used to evaluate the alteration in levels of uterine SEC63 gene expression during the peri-implantation period in mice. Further, both in situ hybridization and immunohistochemical analyses were performed to examine the spatial localization of SEC63 gene expression in mouse uterine tissues. The presence of Sec63 protein in human uterine tissue was also detected by immunohistochemical analysis. Statistical analysis was carried out using Tukey test. Results Uterine SEC63 gene expression was up-regulated and predominantly localized in mouse decidual cells during days 5–8 of pregnancy. More interestingly, Sec63 protein was also detected in human decidua of 10-week pregnancy, whereas was not observed in human endometrial tissues both at proliferative and secretory phases of menstrual cycle. Conclusion The pattern of SEC63 gene expression is consistent with a possible role for SEC63 in decidualization.

ATP stimulates embryo implantation: Decidualization is the process through which the uterine lining is remodeled to enable embryo implantation, which, in mice, depends on the presence of a blastocyst. Gu et al. found that uterine epithelial cells released ATP when a blastocyst was present and that ATP-mediated activation of the purinergic receptor P2Y2 induced decidualization markers and promoted embryo implantation. Experiments in vivo, in primary mouse uterine cells, and in cultured human uterine cells were consistent with ectonucleotidases in the uterine stroma limiting ATP signaling to restrict decidualization and implantation to a short window of time. These findings shed light on the blastocyst-uterine interactions that mediate the establishment of pregnancy. Embryo implantation involves a sterile inflammatory reaction that is required for the invasion of the blastocyst into the decidua. Adenosine triphosphate (ATP) released from stressed or injured cells acts as an important signaling molecule to regulate many key physiological events, including sterile inflammation. We found that the amount of ATP in the uterine luminal fluid of mice increased during the peri-implantation period, and this depended on the presence of an embryo. We further showed that the release of ATP from receptive epithelial cells was likely stimulated by lactate released from the blastocyst through connexin hemichannels. The ATP receptor P2y2 was present on uterine epithelial cells during the preimplantation period and increased in the stromal cells during the time at which decidualization began. Pharmacological inhibition of P2y2 compromised decidualization and implantation. ATP-P2y2 signaling stimulated the phosphorylation of Stat3 in uterine luminal epithelial cells and the expression of early growth response 1 (Egr1) and prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox-2), all of which are required for decidualization and/or implantation, in stromal cells. Short exposure to high concentrations of ATP promoted decidualization of primary stromal cells, but longer exposures or lower ATP concentrations did not. The expression of genes encoding ATP-degrading ectonucleotidases increased in the decidua during the peri-implantation period, suggesting that they may limit the duration of the ATP signal. Together, our results indicate that the blastocyst-induced release of ATP from uterine epithelial cells during the peri-implantation period may be important for the initiation of stromal cell decidualization. [ABSTRACT FROM AUTHOR]

The Notch signaling pathway is required for reproductive success. This pathway activates its transcriptional effector, recombination signal binding protein for immunoglobulin kappa J (Rbpj), to induce transcription of its target genes. This signaling pathway is required for successful decidualization, implantation, and uterine repair following parturition. To identify the compartmental specific roles of the Notch signaling pathway in the establishment of pregnancy, we generated epithelial and decidual stromal cell specific knockouts of Rbpj utilizing lactoferrin iCre and Prl8A2 iCre, respectively. Both conditional knockout mouse models were fertile. The Rbpj epithelial knockout mice displayed 27% resorption sites at E15.5, but this did not significantly impact the number of live born pups compared with controls. In addition, the Rbpj epithelial knockout mice displayed increased estrogen signaling in their stromal compartment. Given that both mouse models exhibited fertility comparable to control animals, the epithelial and stromal specific nature of the iCre recombinases utilized, and previously published Rbpj total uterine knockout mouse models, we conclude that Notch effector Rbpj signaling is required at the initiation of pregnancy to support decidualization in stromal cells, but that Rbpj is not required in the epithelial compartment nor is it required for post-implantation pregnancy success., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High level of uric acid (UA) is the major origin of gout, and is highly associated with various pregnant complications, such as preeclampsia and gestational diabetes. However, UA's level and role in the very early stage of pregnancy has not been uncovered. This study aims to investigate the relevance of serum UA and decidualization, an essential process for the establishment and maintenance of pregnancy in women and mice during the early stage of pregnancy. In this study, we first proved that expression level of UA synthase xanthine dehydrogenase (XDH) is highly increased along with decidualization of endometrial stromal cells in both in vitro and in vivo models. Furthermore, serum and endometrial levels of UA are higher in mice with decidualized uterin horn and in vitro decidualized stromal cells. The existence of monosodium urate (MSU) crystal was also confirmed by immunostaining. Next, the roles of MSU on decidualization were explored by both in vitro and in vivo models. Our data shows MSU crystal but not UA enhances the decidualization response of endometrial stromal cells, via the upregulation of inflammatory genes such Ptgs2 and Il11 . inhibiting of Cox-2 activity abolishes MSU crystal induced higher expression of decidualization marker Prl8a2 . At last, in women, we observed enriched expression of XDH in decidua compare to non-decidualized endometrium, the serum level of UA is significantly increased in women in very early stage of pregnancy, and drop down after elective abortion. In summary, we observed an increased serum UA level in the early stage of women's pregnancy, and proved that the increased level of UA results from the expressed XDH in decidualizing endometrium of both human and mouse, leading to the formation of MSU crystal. MSU crystal can enhance the decidualization response via inflammatory pathways. Our study has uncovered the association between UA, MSU, and decidualization during the early stage of pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhu, Wu, Chen, Kang, Pan, Liu, Li, Yan, Liu, Huang, Yang and Su.)

The endometrium undergoes a pregnancy-delivery-repair cycle multiple times during the reproductive lifespan in females. Decidualization is one of the critical events for the success of this essential process. We have previously reported that Notch1 is essential for artificial decidualization in mice. However, in a natural pregnancy, the deletion of Notch1 (PgrCre/+Notch1f/f, or Notch1d/d) only affects female fertility in the first 30 days of a 6-month fertility test, but not the later stages. In the present study, we undertook a closer evaluation at the first pregnancy of these mice to attempt to understand this puzzling phenomenon. We observed a large number of pregnancy losses in Notch1d/d mice in their first pregnancy, which led to the subfertility observed in the first 30 days of the fertility test. We then demonstrated that the initial pregnancy loss is a consequence of impaired decidualization. Furthermore, we identified a group of genes that contribute to Notch1 regulated decidualization in a natural pregnancy. Gene ontogeny analysis showed that these differentially expressed genes in the natural pregnancy are involved in cell-cell and cell-matrix interactions, different from genes that have been previously identified from the artificial decidualization model, which contribute to cell proliferation and apoptosis. In summary, we determined that Notch1 is essential for normal decidualization in the mouse uterus only in the first pregnancy but not in subsequent ones., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer., Objective: To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis., Setting and Design: The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo., Results: E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model., Conclusions: IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Autoimmune Regulator (AIRE) regulates central immune tolerance by inducing expression of tissue-restricted antigens in thymic medullary epithelial cells, thereby ensuring elimination of autoreactive T cells. Aire mutations in humans and targeted Aire deletion in mice result in multiorgan autoimmune disease, known in humans as autoimmune polyglandular syndrome type 1 (APS-1). APS-1 is characterized by the presence of adrenal insufficiency, chronic mucosal candidiasis, and/or hypoparathyroidism. Additionally, females often present with gonadal insufficiency and infertility. Aire-deficiency (KO) in mice results in oophoritis and age-dependent depletion of follicular reserves. Here, we found that while the majority of young 6-week-old Aire-KO females had normal follicular reserves, mating behavior, and ovulation rates, 50% of females experienced embryonic loss between gestation day (GD) 5.5 and 7.5 that could not be attributed to insufficient progesterone production or decidualization. The quality of GD0.5 embryos recovered from Aire KO mice was reduced, and when cultured in vitro, embryos displayed limited developmental capacity in comparison to those recovered from wild-type (WT) mice. Further, embryos flushed from Aire KO dams at GD3.5 were developmentally delayed in comparison to WT controls and had reduced trophoblastic outgrowth in vitro. We conclude that AIRE does not play a direct role in uterine decidualization. Rather, reduced fertility of Aire-deficient females is likely due to multiple factors, including oophoritis, delayed preimplantation development, and compromised implantation. These effects may be explained by autoimmune targeting of the ovary, embryo, or both. Alternatively, altered embryonic development could be due to a direct role for AIRE in early embryogenesis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Unexplained recurrent pregnancy loss (uRPL) is associated with repeated embryo loss and endometrial repair with elevated endometrial expression of inflammatory cytokines, including IFN-γ. Notch signaling through its transcription factor recombination signal binding protein Jκ (RBPJ) regulates mechanisms including the immune response and repair after tissue injury. Initially, null mutation of RBPJ in the mouse uterus ( Pgr cre/+ Rbpj f/f ; Rbpj c-KO) results in subfertility, but we have found that these mice become infertile after pregnancy as a result of dysfunctional postpartum uterine repair, including delayed endometrial epithelial and myometrial regeneration. RNA sequencing of postpartum uterine repair sites revealed global up-regulation of inflammatory pathways, including IFN signaling. Consistent with elevated IFN-γ, macrophages were recruited and polarized toward an M1-cytotoxic phenotype, which is associated with preventing repair and promoting further tissue injury. Through embryo transfer experiments, we show that dysfunctional postpartum repair directly impairs future embryo implantation in Rbpj c-KO mice. Last, we clinically correlated our findings from the Rbpj c-KO mouse in women diagnosed with uRPL. Reduced RBPJ in women with uRPL was associated with increased levels of IFN-γ. The data, taken together, indicate that RBPJ regulates inflammation during endometrial repair, which is essential for future pregnancy potential, and its dysregulation may serve as an unidentified contributor to uRPL in women.-Strug, M. R., Su, R.-W., Kim, T. H., Mauriello, A., Ticconi, C., Lessey, B. A., Young, S. L., Lim, J. M., Jeong, J.-W., Fazleabas, A. T. RBPJ mediates uterine repair in the mouse and is reduced in women with recurrent pregnancy loss.

Implantation and the establishment of pregnancy are critical for the propagation of the species, but yet remain the limiting steps in human and primate reproduction. Successful implantation requires a competent blastocyst and a receptive endometrium during a specific window of time during the menstrual cycle to initiate the bilateral communication required for the establishment of a successful pregnancy. This chapter provides an overview of these processes and discusses the molecular mechanisms associated with implantation of the blastocyst and decidualization of the uterus in primates.

Background: Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus., Methodology/principal Findings: Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process., Conclusion: Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.

Embryo implantation is a critical step in mammalian reproduction and includes apposition of the blastocyst, attachment to the uterine epithelium and decidualization of the uterine stroma. However, the key mechanism underlying embryo implantation remains to be elucidated. In recent years, high-throughput transcriptomic and proteomic approaches make it possible for studying the expression levels of thousands of genes and proteins simultaneously and thus accelerate the understanding of gene regulatory networks involved in embryo implantation. This review briefly summarizes the advance on mRNA, miRNA and protein expression profile analysis during embryo implantation in human and a variety of animal models.

Background: Delayed implantation is a developmental arrest at the blastocyst stage and a good model for embryo implantation. MicroRNAs (miRNAs) have been shown to be involved in mouse embryo implantation through regulating uterine gene expression. This study was to have an integrative analysis on global miRNA and mRNA expression in mouse uterus under delayed implantation and activation through Illumina sequencing., Methodology/principal Findings: By deep sequencing and analysis, we found that there are 20 miRNAs up-regulated and 42 miRNAs down-regulated at least 1.2 folds, and 268 genes up-regulated and 295 genes down-regulated at least 2 folds under activation compared to delayed implantation, respectively. Many different forms of editing in mature miRNAs are detected. The percentage of editing at positions 4 and 5 of mature miRNAs is significantly higher under delayed implantation than under activation. Although the number of miR-21 reference sequence under activation is slightly lower than that under delayed implantation, the total level of miR-21 under activation is higher than that under delayed implantation. Six novel miRNAs are predicted and confirmed. The target genes of significantly up-regulated miRNAs under activation are significantly enriched., Conclusions: miRNA and mRNA expression patterns are closely related. The target genes of up-regulated miRNAs are significantly enriched. A high level of editing at positions 4 and 5 of mature miRNAs is detected under delayed implantation than under activation. Our data should be valuable for future study on delayed implantation.

Embryo implantation is an intricate interaction between receptive uterus and active blastocyst. The mechanism underlying embryo implantation is still unknown. Although histamine and putrescine are important for embryo implantation and decidualization, excess amount of histamine and putrescine is harmful. Amiloride binding protein 1 (Abp1) is a membrane-associated amine oxidase and mainly metabolizes histamine and putrescine. In this study, we first showed that Abp1 is strongly expressed in the decidua on d 5-8 of pregnancy. Abp1 expression is not detected during pseudopregnancy and under delayed implantation but is detected after estrogen activation. Because Abp1 is mainly localized in the decidua and also strongly expressed during in vitro decidualization, Abp1 might play a role during mouse decidualization. The regulation of estrogen on Abp1 is mediated by transcription factor CCAAT/enhancer-binding protein-β. Abp1 expression is also regulated by cAMP, bone morphogenetic protein 2, and ERK1/2. Abp1 may be essential for mouse embryo implantation and decidualization.

Although Cyclooxygenase-2 (COX-2) is essential for mouse ovulation, fertilization, implantation and decidualization, the regulation and function of COX-2 in rat reproduction are still unknown. This study was designed to examine the action of COX-2 in rat ovulation, implantation and decidualization by using two specific inhibitors of COX-2 (nimesulide and niflumic acid). Compared to control, either nimesulide or niflumic acid significantly inhibited the ovulation in the superovulated rats. Although nimesulide had no obvious effects on the number of implantation sites and the vascular permeability, the expression of PPARdelta, HB-EGF and vimentin proteins was down-regulated in the nimesulide-treated groups. COX-1 protein was upregulated by nimesulide treatment. Nimesulide also had an inhibitory effect on decidualization during early pregnancy and under artificial decidualization. Moreover, nimesulide caused the increase of the gestation period and the reduction of litter size and birth weight compared to controls. Based on our data, rat implantation and decidualization were delayed by nimesulide treatment, resulting in the reduction of litter size and birth weight and the prolongation of gestational length, suggesting that COX-2 plays an important role in implantation and decidualization.