Pregnant Mouse Model Research Articles

Uterine stromal but not epithelial PTGS2 is critical for murine pregnancy success.

Use of non-steroidal anti-inflammatory drugs that target prostaglandin synthase (PTGS) enzymes have been implicated in miscarriage. Further, PTGS2-derived prostaglandins are reduced in the endometrium of patients with a history of implantation failure. However, in the mouse model of pregnancy, peri-implantation PTGS2 function is controversial. Some studies suggest that Ptgs2 -/- mice display deficits in ovulation, fertilization, and implantation, while other studies suggest a role for PTGS2 only in ovulation but not implantation. Further, the uterine cell type responsible for PTGS2 function and role of PTGS2 in regulating implantation chamber formation is not known. To address this we generated tissue-specific deletion models of Ptgs2 . We observed that PTGS2 ablation from the epithelium alone in Ltf cre/+ ; Ptgs2 f/f mice and in both the epithelium and endothelium of the Pax2 cre/+ ; Ptgs2 f/f mice does not affect embryo implantation. Further, deletion of PTGS2 in the ovary, oviduct, and the uterus using Pgr cre/+ ; Ptgs2 f/f does not disrupt pre-implantation events but instead interferes with post-implantation chamber formation, vascular remodeling and decidualization. While all embryos initiate chamber formation, more than half of the embryos fail to transition from blastocyst to epiblast stage, resulting in embryo death and resorbing decidual sites at mid-gestation. Thus, our results suggest no role for uterine epithelial PTGS2 in early pregnancy but instead highlight a role for uterine stromal PTGS2 in modulating post-implantation embryo and implantation chamber growth. Overall, our study provides clarity on the compartment-specific role of PTGS2 and provides a valuable model for further investigating the role of stromal PTGS2 in post-implantation embryo development.

Development of a humanized mouse model with functional human materno-fetal interface immunity.

Materno-fetal immunity possesses specialized characteristics to ensure pathogen clearance while maintaining tolerance to the semiallogeneic fetus. Most of our understanding on human materno-fetal immunity is based on conventional rodent models that may not precisely represent human immunological processes owing to the huge evolutionary divergence. Herein, we developed a pregnant human immune system (HIS) mouse model through busulfan preconditioning, which hosts multilineage human immune subset reconstitution at the materno-fetal interface. Human materno-fetal immunity exhibits a tolerogenic feature at the midgestation stage (embryonic day [E] 14.5), and human immune regulatory subsets were detected in the decidua. However, the immune system switches to an inflammatory profile at the late gestation stage (E19). A cell-cell interaction network contributing to the alternations in the human materno-fetal immune atmosphere was revealed based on single-cell RNA-Seq analysis, wherein human macrophages played crucial roles by secreting several immune regulatory mediators. Furthermore, depletion of Treg cells at E2.5 and E5.5 resulted in severe inflammation and fetus rejection. Collectively, these results demonstrate that the pregnant HIS mouse model permits the development of functional human materno-fetal immunity and offers a tool for human materno-fetal immunity investigation to facilitate drug discovery for reproductive disorders.

Trem2/Syk/PI3K axis contributes to the host protection against Toxoplasma gondii-induced adverse pregnancy outcomes via modulating decidual macrophages.

Decidual macrophages residing at the maternal-fetal interface have been recognized as pivotal factors for maintaining normal pregnancy; however, they are also key target cells of Toxoplasma gondii (T. gondii) in the pathology of T. gondii-induced adverse pregnancy. Trem2, as a functional receptor on macrophage surface, recognizes and binds various kinds of pathogens. The role and underlying mechanism of Trem2 in T. gondii infection remain elusive. In the present study, we found that T. gondii infection downregulated Trem2 expression and that Trem2-/- mice exhibited more severe adverse pregnancy outcomes than wildtype mice. We also demonstrated that T. gondii infection resulted in increased decidual macrophages, which were significantly reduced in the Trem2-/- pregnant mouse model as compared to wildtype control animals. We further described the inhibited proliferation, migration, and invasion functions of trophoblast cell by T. gondii antigens through macrophages as an "intermediate bridge", while this inhibition can be rescued by Trem2 agonist HSP60. Concurrently, Trem2 deficiency in bone marrow-derived macrophages (BMDMs) heightened the inhibitory effect of TgAg on the migration and invasion of trophoblast cells, accompanied by higher pro-inflammatory factors (IL-1β, IL-6 and TNF-α) but a lower chemokine (CXCL1) in T. gondii antigens-treated BMDMs. Furthermore, compelling evidence from animal models and in vitro cell experiments suggests that T. gondii inhibits the Trem2-Syk-PI3K signaling pathway, leading to impaired function of decidual macrophages. Therefore, our findings highlight Trem2 signaling as an essential pathway by which decidual macrophages respond to T. gondii infection, suggesting Trem2 as a crucial sensor of decidual macrophages and potential therapeutic target in the pathology of T. gondii-induced adverse pregnancy.

The effect of Toxoplasma gondii infection on galectin-9 expression in decidual macrophages contributing to dysfunction of decidual NK cells during pregnancy

BackgroundToxoplasma gondii infection causes adverse pregnancy outcomes by affecting the expression of immunotolerant molecules in decidual immune cells. Galectin-9 (Gal-9) is widely expressed in decidual macrophages (dMφ) and is crucial for maintaining normal pregnancy by interacting with the immunomodulatory protein T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3). However, the effects of T. gondii infection on Gal-9 expression in dMφ, and the impact of altered Gal-9 expression levels on the maternal–fetal tolerance function of decidual natural killer (dNK) cells, are still unknown.MethodsPregnancy outcomes of T. gondii-infected C57BL/6 and Lgals9−/− pregnant mice models were recorded. Expression of Gal-9, c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), and Forkhead box protein O1 (FOXO1) was detected by western blotting, flow cytometry or immunofluorescence. The binding of FOXO1 to the promoter of Lgals9 was determined by chromatin immunoprecipitation–polymerase chain reaction (ChIP-PCR). The expression of extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), cAMP-response element binding protein (CREB), phosphorylated CREB (p-CREB), T-box expressed in T cells (T-bet), interleukin 10 (IL-10), and interferon gamma (IFN-γ) in dNK cells was assayed by western blotting.ResultsToxoplasma gondii infection increased the expression of p-JNK and FOXO1 in dMφ, resulting in a reduction in Gal-9 due to the elevated binding of FOXO1 with Lgals9 promoter. Downregulation of Gal-9 enhanced the phosphorylation of ERK, inhibited the expression of p-CREB and IL-10, and promoted the expression of T-bet and IFN-γ in dNK cells. In the mice model, knockout of Lgals9 aggravated adverse pregnancy outcomes caused by T. gondii infection during pregnancy.ConclusionsToxoplasma gondii infection suppressed Gal-9 expression in dMφ by activating the JNK/FOXO1 signaling pathway, and reduction of Gal-9 contributed to dysfunction of dNK via Gal-9/Tim-3 interaction. This study provides new insights for the molecular mechanisms of the adverse pregnancy outcomes caused by T. gondii.Graphical

Maternal High Fat Diet and its Expressions in the Heart and Liver in the Mice Embryogenesis.

The developmental biology for the nonalcoholic fatty liver disease and coronary heart disease are known but elaborative ideas of triglycerides phenomenon in the embryo-genesis of the liver and the heart are still not clear. The aim of the study was to relate different triglycerides like LXRα, LPL, LDL R, PPARG-, SREBP-1C expression in the high fat fed mice with the normal fed diet mice in the process of developmental and embryo-genesis biology. Tissue preparation was done by ripalysis. Different protein content was obtained via western blot for the 6 samples namely a-17.5 days mice embryo heart; b- 0th day or the birthday mice infant heart; c-1 week mice infant heart; d-2 weeks mice infant heart; e-3 weeks mice infant heart; f-Adult mice heart. Protein lysates from the heart tissues of the mice was obtained via homegenization and centrifugation. Hematoxylin and Eosin (H and E) was done to see the fat droplets in the liver tissues at the different developmental stages. LXRα,SREBP-1C expression in 17.5 days mice embryo heart and 0th day or the birthday mice infant heart is highly expressed in the high fat diet. LDL-R in the high fat diet mice is increased in 2 weeks mice infant heart but in17.5 days mice embryo heart and in 0th day or the birthday mice infant heart it is low expression but from 1week mice infant heart to the adult mice heart the expression is in decreasing trend. Similarly LPL is highly expressed in17.5 days mice embryo heart and 1 week mice infant heart and thus low expression in decreasing order until adult mice heart.Thus, these results collectively shows that maternal HF diet increases expression of proteins such as LPL, LDLr in the embryo phase and thus getting normal expressions in the adult phase that facilitate Triglycerides (TAG) hydrolysis across the liver and the heart. Also,maternal high fat diet increases the SREBP1c expression, leading to stimulation of LPL Expression. In summary, using a pregnant mice model, we found that maternal high fat diet increases the fetal fat accumulation. Elevated placental LPL activity and expression of genes that facilitate placental lipid transport suggest that enhanced placental lipid transport may play a key role in maternal nutrition and obesity-induced fetal fat accumulation.

Immunogenicity, safety and dual DIVA-like character of a recombinant candidate vaccine against neosporosis in cattle

Neosporosis is the major infectious cause of abortion and reproductive losses in cattle worldwide; however, there are no available vaccines or drugs to control this disease. Recently, a dual (positive and negative) DIVA-like (Differentiation of Infected from Vaccinated Animals) vaccine was evaluated in a pregnant mouse model of neosporosis, showing promising immunogenic and protective results. The current report aimed to study the safety, the dose-dependent immunogenicity and the dual DIVA-like character of a recombinant subunit vaccine composed of the major surface antigen from Neospora caninum (rNcSAG1) and the carrier/adjuvant Heat shock protein 81.2 from Arabidopsis thaliana (rAtHsp81.2) in cattle. Healthy heifers were separated and assigned to experimental groups A-F and subcutaneously immunized with 2 doses of vaccine formulations 30 days apart as follows: A (n = 4): 50 μg rNcSAG1 + 150 μg rAtHsp81.2; B (n = 4): 200 μg rNcSAG1 + 600 μg rAtHsp81.2; C (n = 4): 500 μg rNcSAG1 + 1,500 μg rAtHsp81.2; D (n = 3): 150 μg rAtHsp81.2; E (n = 3):1,500 μg rAtHsp81.2, and F (n = 3) 2 ml of sterile PBS. The immunization of heifers with the different vaccine or adjuvant doses (groups A-E) was demonstrated to be safe and did not modify the mean value of the evaluated serum biomarkers of metabolic function (GOT/ASP, GPT/ALT, UREA, Glucose and total proteins). The kinetics and magnitude of the immune responses were dose-dependent. The higher dose of the vaccine formulation (group C) stimulated a broad and potent humoral and cellular immune response, characterized by an IgG1/IgG2 isotype profile and IFN-γ secretion. In addition, this was the first time that dual DIVA-like character of a vaccine against neosporosis was demonstrated, allowing us to differentiate vaccinated from infected heifers by two different DIVA compliant test approaches. These results encourage us to evaluate its protective efficacy in infected pregnant cattle in the future.

Toll-like receptor 4 (TLR4) deficiency impedes Toxoplasma gondii excreted-secreted antigens (ESA)-induced abortion

IntroductionToxoplasma gondii is an opportunistic intracellular parasite that is a major pathogenic factor in miscarriage, especially when it occurs early in pregnancy. We have previously demonstrated that the regulation of forkhead box transcription factor (Foxp3) is associated with abortion in early pregnancy caused by excretory-secretory antigen (ESA) of strain China 1. We aimed to reveal the underlying mechanism of miscarriage caused by ESA. MethodsA TLR4−/− pregnant mouse model was successfully constructed. Pregnant mice at gestational day 5 (G5) were injected with ESA. All animals were sacrificed on G13, pregnancy outcomes were observed, and abortion rates were calculated. Placental status observed by Hematoxylin-eosin staining; gene expression was measured by IHC; flow cytometry analysis was used to determine the number and function of regulatory T cells. In EL4 cells, real-time PCR and Western blot were used to evaluate gene expression and cytokines assay. ResultsIn vivo studies revealed that ESA injection caused 83% abortion in pregnant mice but only 35% abortion in TLR4−/− pregnant mice. In addition, ESA attenuated the number and function of regulatory T cells, further suppressed Foxp3, FOXO1 levels, and upregulated CD127 expression. TLR4−/− mice partially reversed this inhibitory effect on regulatory T cells. Furthermore, in vitro studies revealed that ESA inhibited TLR4/NF-κB signaling pathway expression and that TLR4 agonists significantly restored the ESA-induced decrease in Foxp3. DiscussionThese findings suggest that ESA suppresses Foxp3 expression by blocking TLR4/NF-κB signaling, resulting in miscarriage. More importantly, the results indicated that miscarriage caused by ESA is TLR4 dependent.

Long-chain acyl-CoA synthetase-4 regulates endometrial decidualization through a fatty acid β-oxidation pathway rather than lipid droplet accumulation

ObjectiveLipid metabolism plays an important role in early pregnancy, but its effects on decidualization are poorly understood. Fatty acids (FAs) must be esterified by fatty acyl-CoA synthetases to form biologically active acyl-CoA in order to enter the anabolic and/or catabolic pathway. Long-chain acyl-CoA synthetase 4 (ACSL4) is associated with female reproduction. However, whether it is involved in decidualization is unknown. MethodsThe expression of ACSL4 in human and mouse endometrium was detected by immunohistochemistry. ACSL4 levels were regulated by the overexpression of ACSL4 plasmid or ACSL4 siRNA, and the effects of ACSL4 on decidualization markers and morphology of endometrial stromal cells (ESCs) were clarified. A pregnant mouse model was established to determine the effect of ACSL4 on the implantation efficiency of mouse embryos. Modulation of ACSL4 detects lipid anabolism and catabolism. ResultsThrough examining the expression level of ACSL4 in human endometrial tissues during proliferative and secretory phases, we found that ACSL4 was highly expressed during the secretory phase. Knockdown of ACSL4 suppressed decidualization and inhibited the mesenchymal-to-epithelial transition induced by MPA and db-cAMP in ESCs. Further, the knockdown of ACSL4 reduced the efficiency of embryo implantation in pregnant mice. Downregulation of ACSL4 inhibited FA β-oxidation and lipid droplet accumulation during decidualization. Interestingly, pharmacological and genetic inhibition of lipid droplet synthesis did not affect FA β-oxidation and decidualization, while the pharmacological and genetic inhibition of FA β-oxidation increased lipid droplet accumulation and inhibited decidualization. In addition, inhibition of β-oxidation was found to attenuate the promotion of decidualization by the upregulation of ACSL4. The decidualization damage caused by ACSL4 knockdown could be reversed by activating β-oxidation. ConclusionsOur findings suggest that ACSL4 promotes endometrial decidualization by activating the β-oxidation pathway. This study provides interesting insights into our understanding of the mechanisms regulating lipid metabolism during decidualization.

A Review on the Methodology and Use of the Pregnant Mouse Model in the Study of Brucella Reproductive Pathogenesis and Its Abortifacient Effect.

Brucellosis is one of the most common and widespread bacterial zoonoses and is caused by Gram-negative bacteria belonging to the genus Brucella. These organisms are able to infect and replicate within the placenta, resulting in abortion, one of the main clinical signs of brucellosis. Although the mouse model is widely used to study Brucella virulence and, more recently, to evaluate the protection of new vaccines, there is no clear consensus on the experimental conditions (e.g., mouse strains, doses, routes of inoculation, infection/pregnancy time) and the natural host reproducibility of the pregnant mouse model for reproductive brucellosis. This lack of consensus calls for a review that integrates the major findings regarding the effect of Brucella wild-type and vaccine strains infections on mouse pregnancy. We found sufficient evidence on the utility of the pregnant mouse model to study Brucella-induced placentitis and abortion and propose suitable experimental conditions (dose, time of infection) and pregnancy outcome readouts for B. abortus and B. melitensis studies. Finally, we discuss the utility and limitations of the pregnant mouse as a predictive model for the abortifacient effect of live Brucella vaccines.

Di-n-butyl phthalate induces toxicity in male fetal mouse testicular development by regulating the MAPK signaling pathway

“White pollution” has a significant impact on male reproduction. Di-n-butyl phthalate (DBP) is one of the most important factors in this type of pollution. Currently, research from international sources has demonstrated the significant reproductive toxicity of DBP. However, most of these studies have focused mainly on hormones expression at the protein and mRNA levels and the specific molecular targets of DBP and its mechanisms of action remain unclear. In this study, we established a Sprague Dawley pregnant mouse model exposed to DBP, and all male offspring were immediately euthanized at birth and bilateral testes were collected. We found through transcriptome sequencing that cell apoptosis and MAPK signaling pathway are the main potential pathways for DBP induced reproductive toxicity. Molecular biology analyses revealed a significant increase in the protein levels of JNK1(MAPK8) and BAX, as well as a significant increase in the BAX/BCL2 ratio after DBP exposure. Therefore, we propose that DBP induces reproductive toxicity by regulating JNK1 expression to activate the MAPK signaling pathway and induce reproductive cell apoptosis. In conclusion, our study provides the first evidence that the MAPK signaling pathway is involved in DBP-induced reproductive toxicity and highlights the importance of JNK1 as a potential target of DBP in inducing reproductive toxicity.

Epigenetic regulation by KDM5A mediates the effects of prenatal PM2.5 exposure on hippocampal development and synaptic integrity through the Shh signaling pathway

Prenatal environmental exposure could be an essential health risk factor associated with neurodevelopmental disorders in offspring. However, the exact mechanisms underlying the impact of prenatal PM2.5 exposure on offspring cognition remain unclear. In our recent study using a PM2.5 exposed pregnant mouse model, we observed significant synaptic dysfunction in the hippocampi of the offspring. Concurrently, the epigenetic regulator of KDM5A and the Shh signaling pathway exhibited decreased activities. Significantly, changes in hippocampal KDM5A and Shh levels directly correlated with PM2.5 exposure intensity. Subsequent experiments revealed a marked reduction in the expression of Shh signaling and related synaptic proteins when KDM5A was silenced in cells. Notably, the effects of KDM5A deficiency were reversed significantly with the supplementation of a Shh activator. Furthermore, our findings indicate that Shh activation significantly attenuates PM2.5-induced synaptic impairments in hippocampal neurons. We further demonstrated that EGR1, a transcriptional inhibitor, plays a direct role in KDM5A’s regulation of the Shh pathway under conditions of PM2.5 exposure. Our results suggest that the KDM5A’s inhibitory regulation on the Shh pathway through the EGR1 gene is a crucial epigenetic mechanism underlying the synaptic dysfunction in hippocampal neurons caused by maternal PM2.5 exposure. This emphasizes the role of epigenetic regulations in neurodevelopmental disorders caused by environmental factors.

B(a)P induces ovarian granulosa cell apoptosis via TRAF2-NFκB-Caspase1 axis during early pregnancy

Benzo(a)pyrene [B(a)P] is an environmental endocrine disruptor with reproductive toxicity. The corpus luteum (CL) of the ovary plays an important role in embryo implantation and pregnancy maintenance. Our previous studies have shown that B(a)P exposure affects embryo implantation and endometrial decidualization in mouse, but its effects and mechanisms on CL function remain unclear. In this study, we explore the mechanism of ovarian toxicity of B(a)P using a pregnant mouse model and an in vitro model of human ovarian granulosa cells (GCs) KGN. Pregnant mice were gavaged with corn oil or 0.2 mg/kg.bw B(a)P from pregnant day 1 (D1) to D7, while KGN cells were treated with DMSO, 1.0IU/mL hCG, or 1.0IU/mL hCG plus benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), a B(a)P metabolite. Our findings revealed that B(a)P exposure damaged embryo implantation and reduced estrogen and progesterone levels in early pregnant mice. Additionally, in vitro, BPDE impaired luteinization in KGN cells. We observed that B(a)P/BPDE promoted oxidative stress (OS) and inflammation, leading to apoptosis rather than pyroptosis in ovaries and luteinized KGN cells. This apoptotic response was mediated by the activation of inflammatory Caspase1 through the cleavage of BID. Furthermore, B(a)P/BPDE inhibited TRAF2 expression and suppressed NFκB signaling pathway activation. The administration of VX-765 to inhibit the Caspase1 activation, over-expression of TRAF2 using TRAF2-pcDNA3.1 (+) plasmid, and BetA-induced activation of NFκB signaling pathway successfully alleviated BPDE-induced apoptosis and cellular dysfunction in luteinized KGN cells. These findings were further confirmed in the KGN cell treated with H2O2 and NAC. In conclusion, this study elucidated that B(a)P/BPDE induces apoptosis rather than pyroptosis in GCs via TRAF2-NFκB-Caspase1 during early pregnancy, and highlighting OS as the primary contributor to B(a)P/BPDE-induced ovarian toxicity. Our results unveil a novel role of TRAF2-NFκB-Caspase1 in B(a)P-induced apoptosis and broaden the understanding of mechanisms underlying unexplained luteal phase deficiency.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage

BackgroundWith rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored.ResultsIn this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model.ConclusionsExposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.Graphical

Cycasin derivative: a potential embryotoxic component of Atractylodes macrocephala rhizome for limb malformation.

The rhizome of Atractylodes macrocephala Koidz. (Asteraceae), called Atractylodes macrocephala rhizome (AMR) and known by its traditional name Bai Zhu, is a prominent Chinese herbal medicine employed for preventing miscarriage. However, our previous study revealed that high dosages of AMR administered during pregnancy could cause embryotoxicity but the specific embryotoxic components and their underlying mechanisms remain unclear. This study aimed to screen and identify the potential embryotoxic components of AMR. The AMR extracts and sub-fractions were analyzed by thin layer chromatography and subsequently screened by in vitro mouse limb bud micromass and mouse whole embryo culture bioassays. The embryotoxic fractions from AMR were further evaluated in vivo using a pregnant mouse model. The structures of the potential embryotoxic components were analyzed using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). In vitro and in vivo bioassays revealed that AMR glycoside-enriched sub-fractions (AMR-A-IIa and AMR-A-IIb) exhibited potential embryotoxicity. These sub-fractions, when administered to pregnant animals, increased the incidence of stillbirth and congenital limb malformations. MS spectrometry analysis identified cycasin derivatives in both sub-fractions, suggesting their possible role in the observed limb malformations. However, further experiments are necessary to validate this hypothesis and to elucidate the underlying mechanisms. Our study provides significant scientific evidence on the pharmacotoxicity of AMR, which is important for the safe clinical application of commonly used Chinese herbal medicines during pregnancy.

Zika purified inactivated virus (ZPIV) vaccine reduced vertical transmission in pregnant immunocompetent mice

Zika virus (ZIKV) is a significant threat to pregnant women and their fetuses as it can cause severe birth defects and congenital neurodevelopmental disorders, referred to as congenital Zika syndrome (CZS). Thus, a safe and effective ZIKV vaccine for pregnant women to prevent in utero ZIKV infection is of utmost importance. Murine models of ZIKV infection are limited by the fact that immunocompetent mice are resistant to ZIKV infection. As such, interferon-deficient mice have been used in some preclinical studies to test the efficacy of ZIKV vaccine candidates against lethal virus challenge. However, interferon-deficient mouse models have limitations in assessing the immunogenicity of vaccines, necessitating the use of immunocompetent mouse pregnancy models. Using the human stat2 knock-in (hSTAT2KI) mouse pregnancy model, we show that vaccination with a purified formalin-inactivated Zika virus (ZPIV) vaccine prior to pregnancy successfully prevented vertical transmission. In addition, maternal immunity protected offspring against postnatal challenge for up to 28 days. Furthermore, passive transfer of human IgG purified from hyper-immune sera of ZPIV vaccinees prevented maternal and fetal ZIKV infection, providing strong evidence that the neutralizing antibody response may serve as a meaningful correlate of protection.

MiR-124-3p negatively impacts embryo implantation via suppressing uterine receptivity formation and embryo development

During embryo implantation, blastocyst interacts with the receptivity endometrium and the endometrial epithelium secretes nurturing fluid to support embryonic development. Interferon-λ (IFN-λ) is a novel, non-redundant regulator that participates in the fetal–maternal interaction; however, the precise molecular mechanism underlying its impact on uterine receptivity remains elusive. Here, microarray profiling revealed that 149 specific miRNAs were differentially expressed in the human endometrial cells following IFN-λ treatment. In particular, miR-124-3p expression was significantly reduced after IFN-λ treatment (p < 0.05). An in vivo mouse pregnancy model showed that miR-124-3p overexpression notably decreased embryo implantation rate and led to an aberrant epithelial phenotype. Furthermore, miR-124-3p negatively impacted the migration and proliferation of endometrial cells, and hindered embryonic developmental competence in terms of blastocyst formation and global DNA re-methylation. Downstream analysis showed that LIF, MUC1 and BCL2 are potential target genes for miR-124-3p, which was confirmed using western blotting and immunofluorescence assays. In conclusion, IFN-λ-driven downregulation of miR-124-3p during embryo implantation modulates uterine receptivity. The dual functional role of miR-124-3p suggests a cross-talk model wherein, maternal endometrial miRNA acts as a transcriptomic modifier of the peri-implantation endometrium and embryo development.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy

Objective This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. Methods Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. Results Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). Conclusions The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Gestational exposure to 1-NP induces ferroptosis in placental trophoblasts via CYP1B1/ERK signaling pathway leading to fetal growth restriction

Fetal growth restriction (FGR) is a prevalent complication in obstetrics, yet its exact aetiology remains unknown. Numerous studies suggest that the degradation of the living environment is a significant risk factor for FGR. 1-Nitropyrene (1-NP) is a widespread environmental pollutant as a representative substance of nitro-polycyclic aromatic hydrocarbons. In this study, we revealed that 1-NP induced FGR in fetal mice by constructing 1-NP exposed pregnant mice models. Intriguingly, we found that placental trophoblasts of 1-NP exposed mice exhibited significant ferroptosis, which was similarly detected in placental trophoblasts from human FGR patients. In this regard, we established a 1-NP exposed cell model in vitro using two human trophoblast cell lines, HTR8/SVneo and JEG-3. We found that 1-NP not only impaired the proliferation, migration, invasion and angiogenesis of trophoblasts, but also induced severe cellular ferroptosis. Meanwhile, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively rescued 1-NP-induced trophoblast biological function impairment. Mechanistically, we revealed that 1-NP regulated ferroptosis by activating the ERK signaling pathway. Moreover, we innovatively revealed that CYP1B1 was essential for the activation of ERK signaling pathway induced by 1-NP. Overall, our study innovatively identified ferroptosis as a significant contributor to 1-NP induced trophoblastic functional impairment leading to FGR and clarified the specific mechanism by which 1-NP induced ferroptosis via the CYP1B1/ERK signaling pathway. Our study provided novel insights into the aetiology of FGR and revealed new mechanisms of reproductive toxicity of environmental pollutants.

Melatonin alleviates the toxic effect of di(2-ethylhexyl) phthalate on oocyte quality resulting from CEBPB suppression during primordial follicle formation

Presently, the exposure of plasticizers to humans and animals occurs daily, which pose a potential threat to reproductive health. In the present study, a pregnant mouse model exposed to di(2-ethylhexyl) phthalate (DEHP, one of the most common plasticizers) and melatonin was established, and the single-cell transcriptome technology was applied to investigate the effects of melatonin in ovarian cells against DEHP. Results showed that DEHP markedly altered the gene expression pattern of ovarian cells, and severely weakened the histone methylation modification of oocytes. The administration of melatonin recovered the expression of LHX8 and SOHLH1 proteins that essential for primordial follicle formation, and increased the expression of CEBPB, as well as key genes of histone methylation modification (such as Smyd3 and Kdm5a). In addition, the ovarian damage caused by DEHP was also relieved after the overexpression of CEBPB, which suggested melatonin could improve primordial follicle formation progress via enhancing CEBPB expression in mice. Besides, the apoptosis of ovarian cells induced by DEHP also was diminished by melatonin. The study provides evidence of melatonin preventing the damage mediated by plasticizers on the reproductive system in females and CEBPB may serve as a downstream target factor of melatonin in the process.

KLF4 down-regulation underlies placental angiogenesis impairment induced by maternal glucose intolerance in late pregnancy

Maternal glucose intolerance in late pregnancy can easily impair pregnancy outcomes and placental development. The impairment of placental angiogenesis is closely related to the occurrence of glucose intolerance during pregnancy, but the mechanism remains largely unknown. In this study, the pregnant mouse model of maternal high-fat diet and endothelial injury model of porcine vascular endothelial cells (PVECs) was used to investigate the effect of glucose intolerance on pregnancy outcomes and placental development. Feeding pregnant mice, a high-fat diet was shown to induce glucose intolerance in late pregnancy, and significantly increase the incidence of resorbed fetuses. Moreover, a decrease was observed in the proportion of blood sinusoids area and the expression level of CD31 in placenta, indicating that placental vascular development was impaired by high-fat diet. Considering that hyperglycemia is an important symptom of glucose intolerance, we exposed PVECs to high glucose (50 mM), which verified the negative effects of high glucose on endothelial function. Bioinformatics analysis further emphasized that high glucose exposure could significantly affect the angiogenesis-related functions of PVECs and predicted that Krüppel-like factor 4 (KLF4) may be a key mediator of these functional changes. The subsequent regulation of KLF4 expression confirmed that the inhibition of KLF4 expression was an important reason why high glucose impaired the endothelial function and angiogenesis of PVECs. These results indicate that high-fat diet can aggravate maternal glucose intolerance and damage pregnancy outcome and placental angiogenesis, and that regulating the expression of KLF4 may be a potential therapeutic strategy for maintaining normal placental angiogenesis.