Pregnancy Outcomes In Mice Research Articles

Subacute exposure to DEHP leads to impaired decidual reaction and exacerbates the risk of early miscarriage in mice.

To investigate the effect of subacute exposure of Di (2-ethylhexyl) phthalate (DEHP) on endometrial decidualization and early pregnancy miscarriage in mice. CD1 mice were orally administrated with 300 (low-dose group), 1000 (medium-dose group), or 3000 mg·kg－1·d－1 DEHP (1/10 LD50, high-dose group) for 28 days, respectively. An early natural pregnancy model and an artificially induced decidualization model were established. The uterine tissues were collected on D7 of natural pregnancy and D8 of artificially induced decidualization, respectively. The effects of a subacute exposure to DEHP on the decidualization of mice were detected by HE staining, Masson staining, TUNEL assay, and Western blotting. A model of spontaneous abortion was constructed in mice after subacute exposure to 300 mg·kg－1·d－1 DEHP, and the effect of impaired decidualization on pregnancy was investigated by observing the pregnancy outcome on the 10th day of gestation. Compared with the control group, the conception rate was significantly decreased in the high-dose DEHP subacute exposure group (P<0.05). HE staining showed that, compared with the control group, the decidual stromal cells in the low- and medium-dose exposure groups were disorganized, the nuclei of the cells were irregular, the cytoplasmic staining was uneven, and the number of polymorphonuclear cells was significantly reduced. Masson staining showed that compared with the control group, the collagen fibers in the decidua region of the DEHP low-dose group and the medium-dose group were more distributed, more abundant and more disorderly. TUNEL assay showed increased apoptosis in the decidua area compared to the control group. Western blotting showed that the expression of BMP2, a marker molecule for endometrial decidualization, was significantly reduced (P<0.05 or P<0.01). The abortion rate and embryo resorption rate were increased, and the number of embryos, uterine wet weight, uterine area and placenta wet weight were decreased in DEHP low-dose group compared to the control group stimulated by mifepristone, an abortifacient drug (P<0.05 or P<0.01). Subacute exposure to DEHP leads to impaired endometrial decidualization during early pregnancy and exacerbates the risk of adverse pregnancy outcomes in mice.

The improvement of inflammatory infiltration and pregnancy outcome in mice with recurrent spontaneous abortion by human amniotic mesenchymal stem cells.

Recurrent spontaneous abortion is thought to be mostly triggered by immune-related causes. Mesenchymal stem cells, which exhibit the traits of multi-directional differentiation capacity and low immunogenicity, have recently been recommended as a viable treatment for spontaneous abortion-prone mice to increase the success of pregnancy. Amniotic membrane tissue is a byproduct of pregnancy and delivery that has a wide range of potential uses due to its easy access to raw materials and little ethical constraints. To construct an abortion-prone mouse model for this investigation, CBA/J female mice were coupled with male DBA/2 mice, while CBA/J female mice were paired with male BALB/c mice as a control. The identical volume of human amniotic mesenchymal stem cells or phosphate buffer was injected intraperitoneally on the 4.5th day of pregnancy. CBA/J female mice were sacrificed by cervical dislocation on the 13.5th day of pregnancy, the embryo absorption rate was calculated, and the uterus, decidua tissues and placenta were gathered for examination. Through detection, it was discovered that human amniotic mesenchymal stem cells significantly increased the expression of interleukin 10 and transforming growth factor beta, while they significantly decreased the expression of interleukin 1 beta and interleukin 6, improved vascular formation and angiogenesis, and minimized the embryo absorption rate and inflammatory cell infiltration in the recurrent spontaneous abortion + human amniotic mesenchymal stem cells group. In any case, human amniotic mesenchymal stem cells regulate inflammatory factors and cell balance at the maternal-fetal interface, which result in a reduction in the rate of embryo absorption and inflammatory infiltration and provide an innovative perspective to the clinical therapy of recurrent spontaneous abortion.

Increased miR-3074-5p expression promotes M1 polarization and pyroptosis of macrophages via ERα/NLRP3 pathway and induces adverse pregnancy outcomes in mice

Decidual macrophages (dMϕs) play critical roles in regulation of immune-microhomeostasis at maternal-fetal interface during pregnancy, but the underlying molecular mechanisms are still unclear. In this study, it was found that litter size and fetal weight were significantly reduced, whereas the rate of embryo resorption was increased in miR-3074-5p knock-in (3074-KI) pregnant mice, compared to that of wild-type (WT) pregnant mice. Plasma levels of pro-inflammatory cytokines in 3074-KI pregnant mice were also significantly elevated compared to WT pregnant mice at GD7.5. The quantity of M1-Mϕs in uterine tissues of 3074-KI pregnant mice was significantly increased compared to WT pregnant mice at GD13.5. Estrogen receptor-α (ERα) was validated to be a target of miR-3074-5p. Either miR-3074-5p overexpression or ERα knockdown promoted transcriptional activity of NF-κB/p65, induced M1-polarization and pyroptosis of THP1-derived Mϕs, accompanied with increased intracellular levels of cleaved Caspase-1, cleaved IL-1β, NLRP3, cleaved GSDMD and ASC aggregation. Furthermore, ERα could not only bind to NLRP3 or ASC directly, but also inhibit the interaction between NLRP3 and ASC. The endometrial miR-3074-5p expression level at the middle secretory stage of repeated implantation failure (RIF) patients was significantly decreased compared to that of control fertile women. These data indicated that miR-3074-5p could promote M1 polarization and pyroptosis of Mϕs via activation of NLRP3 inflammasome by targeting ERα, and the dysregulation of miR-3074-5p expression in dMϕs might damage the embryo implantation and placentation by interfering with inflammatory microenvironment at the maternal-fetal interface during early pregnancy.

Obesity and diet independently affect maternal immunity, maternal gut microbiota and pregnancy outcome in mice.

Maternal obesity poses risks for both mother and offspring during pregnancy, with underlying mechanisms remaining largely unexplored. Obesity is associated with microbial gut dysbiosis and low-grade inflammation, and also the diet has a major impact on these parameters. This study aimed to investigate how maternal obesity and diet contribute to changes in immune responses, exploring potential associations with gut microbiota dysbiosis and adverse pregnancy outcomes in mice. Before mating, C57BL/6 mice were assigned to either a high-fat-diet (HFD) or low-fat-diet (LFD) to obtain obese (n=17) and lean (n=10) mice. To distinguish between the effects of obesity and diet, 7 obese mice were switched from the HFD to the LFD from day 7 until day 18 of pregnancy ("switch group"), which was the endpoint of the study. T helper (Th) cell subsets were studied in the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP), while monocyte subsets and activation status were determined in maternal blood (flow cytometry). Feces were collected before and during pregnancy (day 7,14,18) for microbiota analysis (16S rRNA sequencing). Pregnancy outcome included determination of fetal and placental weight. Obesity increased splenic Th1 and regulatory T cells, MLN Th1 and PP Th17 cells and enhanced IFN-γ and IL-17A production by splenic Th cells upon ex vivo stimulation. Switching diet decreased splenic and PP Th2 cells and classical monocytes, increased intermediate monocytes and activation of intermediate/nonclassical monocytes. Obesity and diet independently induced changes in the gut microbiota. Various bacterial genera were increased or decreased by obesity or the diet switch. These changes correlated with the immunological changes. Fetal weight was lower in the obese than the lean group, while placental weight was lower in the switch than the obese group. This study demonstrates that obesity and diet independently impact peripheral and intestinal immune responses at the end of pregnancy. Simultaneously, both factors affect specific bacterial gut genera and lead to reduced fetal or placental weight. Our data suggest that switching diet during pregnancy to improve maternal health is not advisable and it supports pre/probiotic treatment of maternal obesity-induced gut dysbiosis to improve maternal immune responses and pregnancy outcome.

Subunit nanovaccine elicited T cell functional activation controls Trypanosoma cruzi mediated maternal and placental tissue damage and improves pregnancy outcomes in mice

This study investigated a candidate vaccine effect against maternal Trypanosoma cruzi (Tc) infection and improved pregnancy outcomes. For this, TcG2 and TcG4 were cloned in a nanoplasmid optimized for delivery, antigen expression, and regulatory compliance (nano2/4 vaccine). Female C57BL/6 mice were immunized with nano2/4, infected (Tc SylvioX10), and mated 7-days post-infection to enable fetal development during the maternal acute parasitemia phase. Females were euthanized at E12–E17 (gestation) days. Splenic and placental T-cell responses were monitored by flow cytometry. Maternal and placental/fetal tissues were examined for parasites by qPCR and inflammatory infiltrate by histology. Controls included age/immunization-matched non-pregnant females. Nano2/4 exhibited no toxicity and elicited protective IgG2a/IgG1 response in mice. Nano2/4 signaled a splenic expansion of functionally active CD4+ effector/effector memory (Tem) and central memory (Tcm) cells in pregnant mice. Upon challenge infection, nano2/4 increased the splenic CD4+ and CD8+T cells in all mice and increased the proliferation of CD4+Tem, CD4+Tcm, and CD8+Tcm subsets producing IFNγ and cytolytic molecules (PRF1, GZB) in pregnant mice. A balanced serum cytokines/chemokines response and placental immune characteristics indicated that pregnancy prevented the overwhelming damaging immune response in mice. Importantly, pregnancy itself resulted in a significant reduction of parasites in maternal and fetal tissues. Nano2/4 was effective in arresting the Tc-induced tissue inflammatory infiltrate, necrosis, and fibrosis in maternal and placental tissues and improving maternal fertility, placental efficiency, and fetal survival. In conclusion, we show that maternal nano2/4 vaccination is beneficial in controlling the adverse effects of Tc infection on maternal health, fetal survival, and pregnancy outcomes.

Administration of Akebia Saponin D Improved Blood Lipid Levels and Pregnancy Outcomes in Mice with Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM) is a prevalent and severe metabolic disease in pregnant women that is characterized by a high incidence. Placental oxidative stress and inflammation are recognized as the primary contributors to GDM pathogenesis. The repressive effect of akebia saponin D (ASD) on oxidative stress and inflammation has been demonstrated in various diseases. To investigate the impact of ASD on GDM. Animal experimental study. GDM mice were intraperitoneally treated with ASD. The effect of ASD on GDM symptoms, blood lipid levels, pancreatic tissue damage, gestational outcomes, oxidative stress, and inflammation was assessed via intraperitoneal glucose and insulin tolerance tests, serum glucose and insulin level determination, lipid biochemistry analysis, pathological staining, oxidative stress evaluation, western blot analysis, and enzyme-linked immunosorbent assay. ASD reduced the GDM-induced increase in body weight and blood glucose levels while restoring the decreased insulin levels associated with GDM. In addition, ASD improved the serum lipid parameters, pancreatic tissue damage, and gestational outcomes in GDM mice. Furthermore, ASD reversed the decreased levels of superoxide dismutase and glutathione while reducing the elevated concentrations of malondialdehyde and myeloperoxidase in GDM mice. In addition, ASD rescued the relative protein expression of nuclear factor-E2-related factor 2 and heme oxygenase-1 in the placenta of GDM mice. Additionally, ASD counteracted the increase in tumor necrosis factor-α, interleukin (IL)-6, and IL-1β levels in the sera and placenta of GDM mice. ASD suppressed oxidative stress and inflammation to effectively relieve symptoms and gestational outcomes of the GDM mice.

A novel therapeutic approach for endometriosis using adipose-derived stem cell-derived conditioned medium- A new hope for endometriotic patients in improving fertility.

Endometriosis is defined as the growth of endometrial glands and stromal cells in a heterotopic location with immune dysregulation. It usually leads to chronic pelvic pain and subfertility. Although various treatments are available, the recurrence rate remains high. Adipose tissue is an abundant source of multipotent mesenchymal adipose-derived stem cells (ADSCs). ADSCs display effects on not only tissue regeneration, but also immune regulation. Thus, the current study aims to test the effects of ADSCs on the growth of endometriosis. ADSCs isolated from lipoaspiration-generated adipose tissue and their conditioned medium (ADSC-CM) were subjected to quality validation, including karyotyping as well as growth promotion and sterility tests for microbial contamination under Good Tissue Practice and Good Manufacturing Practice regulations. An autologous endometriosis mouse model was established by suturing endometrial tissue to peritoneal wall followed by treating with DMEM/F12 medium, ADSC-CM, ADSCs or ADSC-CM+ADSCs for 28 days. The area of endometriotic cysts and the degree of pelvic adhesion were measured. ICAM-1, VEGF and caspase 3 expression was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. Moreover, the mice were allowed to mate and deliver. The pregnancy outcomes were recorded. The ADSC-CM was subjected to proteomics analysis with further data mining with Ingenuity Pathway Analysis (IPA). Both ADSC-CM and ADSCs passed quality validation. ADSC-CM reduced the area of endometriotic cysts. The inhibition by ADSC-CM was obliterated by adding ADSCs. The presence of ADSCs with or without ADSC-CM increased the peritoneal adhesion. ADSC-CM inhibited ICAM-1 and VEGF mRNA and protein expression, whereas the addition of ADSCs not only did not inhibit by itself, but also blocked the inhibition by ADSC-CM. The resorption rate was reduced by ADSC-CM. The number of live birth/dam and the survival rate of pup at 1 week-old were both increased by ADSC-CM in mice with endometriosis. IPA demonstrated that PTX3 was potentially critical for the inhibition of endometriosis by ADSC-CM due to its anti-inflammatory and antiangiogenic properties as well as its importance in implantation. ADSC-CM inhibited endometriosis development and improved pregnancy outcomes in mice. Potential translation to clinical treatment for human endometriosis is expected.

Effects of Acute and Chronic Trypanosoma cruzi Infection on Pregnancy Outcomes in Mice: Parasite Transmission, Mortality, Delayed Growth, and Organ Damage in Pups

Chagas disease is caused by Trypanosoma cruzi. This study aimed to determine the effects of T. cruzi infection on fertility rate and health of the newborn pups in pregnant mice. Female mice were challenged with T. cruzi and mated at 21 days (acute parasitemic phase) or 90 days (chronic parasite persistence phase) after infection. Pups were examined for growth up to 20 days after birth; and parasite burden in brain, heart, skeletal muscle, and intestine was measured by real-time quantitative PCR. The inflammatory infiltrate, necrosis, and fibrosis in pups' heart and brain tissues were evaluated by histology. T. cruzi infection in dams delayed the onset of pregnancy, decreased the fertility rate, and led to vertical transmission of parasite to the pups. Furthermore, infected dams delivered pups that exhibited decreased survival rate, decreased birth weight, and decreased growth rate. Significantly increased inflammation, necrosis, and fibrosis of cardiac and brain tissues were noted in pups born to infected dams. Initial challenge with higher parasite dose had more detrimental effects on fertility rate and pups' health in both acutely and chronically infected dams. In conclusion, mice offer a promising model to evaluate the efficacy of new vaccines and therapeutic drugs in controlling the acute and chronic maternal T. cruzi infection and congenital transmission to newborns, and in improving the fertility rate and pups' health outcomes.

Screening of Organophosphate Flame Retardants with Placentation-Disrupting Effects in Human Trophoblast Organoid Model and Characterization of Adverse Pregnancy Outcomes in Mice.

Background:Abnormal placental development may result in adverse pregnancy outcomes and metabolic diseases in adulthood; however, it remains unknown whether and how xenobiotics affect human placentation.Objectives:This study aimed to screen and identify placentation-disrupting chemicals in commonly used organophosphate flame retardants (OPFRs) and, if identified, to investigate potential adverse effects on placentation in relation to adverse pregnancy outcomes and metabolic disorder in offspring in mice.Methods:We devised a high-throughput immunofluorescence screening assay based on human trophoblast organoids and used it to screen OPFRs that inhibit the proliferation of organoids. One identified chemical was assessed for its effects on placentation by evaluating villous cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts using immunofluorescence and a mitochondrial stress test after 2 d of exposure. A 10-d exposure study was further performed to observe the dynamic effect of the OPFR on the structure of the organoids. RNA-sequencing and western blotting experiments were performed to explore the associated pathways, and a potential binding protein was identified by immunoprecipitation and in vitro kinase activity assays. Animal studies were performed to determine whether the findings in organoids could be replicated in mice and to observe adverse pregnancy outcomes.Results:The proliferation of organoids exposed to three aryl-OPFRs was significantly lower than the proliferation of control organoids. Further analysis demonstrated that one such chemical, 2-ethylhexyl-diphenyl phosphate (EHDPP), disrupted placentation in organoids. Mechanistically, EHDPP interfered with insulin-like growth factor 1 receptor (IGF1R) to inhibit aerobic respiration. Mice exposed to EHDPP at a physiological human concentrations exhibited immature and mature placental disorders, which correlated with fetal growth restriction, implantation failure, stillbirth, and impaired glucose tolerance.Conclusions:The human trophoblast organoid model showed that the commonly used OPFRs disrupted placentation via IGF1R, indicating that its use may contribute to adverse pregnancy outcomes and metabolic disorders in offspring. https://doi.org/10.1289/EHP10273

Subacute Exposure to an Environmentally Relevant Dose of Di-(2-ethylhexyl) Phthalate during Gestation Alters the Cecal Microbiome, but Not Pregnancy Outcomes in Mice

Di-2-ethylhexyl phthalate (DEHP) is a plasticizer commonly found in polyvinyl chloride, medical equipment, and food packaging. DEHP has been shown to target the reproductive system and alter the gut microbiome in humans and experimental animals. However, very little is known about the impact of DEHP-induced microbiome changes and its effects during pregnancy. Thus, the objective of this study was to investigate the effects of DEHP exposure during pregnancy on the cecal microbiome and pregnancy outcomes. Specifically, this study tested the hypothesis that subacute exposure to DEHP during pregnancy alters the cecal microbiome in pregnant mice, leading to changes in birth outcomes. To test this hypothesis, pregnant dams were orally exposed to corn oil vehicle or 20 µg/kg/day DEHP for 10 days and euthanized 21 days after their last dose. Cecal contents were collected for 16S Illumina and shotgun metagenomic sequencing. Fertility studies were also conducted to examine whether DEHP exposure impacted birth outcomes. Subacute exposure to environmentally relevant doses of DEHP in pregnant dams significantly increased alpha diversity and significantly altered beta diversity. Furthermore, DEHP exposure during pregnancy significantly increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Deferribacteres compared with controls. The affected taxonomic families included Deferribacteraceae, Lachnospiraceae, and Mucisprillum. In addition to changes in the gut microbiota, DEHP exposure significantly altered 14 functional pathways compared with the control. Finally, DEHP exposure did not significantly impact the fertility and birth outcomes compared with the control. Collectively, these data indicate that DEHP exposure during pregnancy shifts the cecal microbiome, but the shifts do not impact fertility and birth outcomes.

Site specificity of blastocyst hatching significantly influences pregnancy outcomes in mice.

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P<.05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P<.05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P<.05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P<.05) at 2days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

Pregnancy Outcome in Mice with Protein S Complete Deficiency

Introduction:Pregnancy is associated with a shift of the coagulation balance with increased concentration of procoagulant factors, decreased concentration of some anticoagulants and diminished fibrinolytic activity. These changes provoke a state of hypercoagulability that protects pregnant women from fatal hemorrhage during delivery but predispose them to thromboembolism.Protein S (PS) is a natural anticoagulant that acts as a cofactor of activated protein C and tissue factor pathway inhibitor. PS complete deficiency leads to life threatening skin necrosis and disseminated intravascular coagulation (DIC) named purpura fulminans (PF) that leads to death if untreated. Pregnant women with partial PS deficiency are at high risk of late fetal loss. The beneficial role of thromboprophylaxis in ameliorating pregnancy outcome in patients with inherited thrombophilia is debated.Here, we used mice with complete PS deficiency (Pros1-/-) fully rescued from lethality by the lack of factor VIII (F8-/-Pros1-/-). Characterization of F8-/-Pros1-/- mice revealed no signs of PF or DIC. These mice were used to investigate the effect of PS complete deficiency on pregnancy.Methods:Timed matings were set to generate E9-E17.5 embryos from F8-/-Pros1-/-, F8-/-Pros1+/+ and F8+/+Pros1+/+ pregnant mice (n=3-9/genotype). Embryos viability and genotype were evaluated. At the time of sacrifice, blood was drawn from pregnant female to evaluate blood cells count profile and coagulation parameters (fibrinogen, thrombin anti-thrombin complex; TAT) were measured. Anti-fibrin immunostaining was performed in collected embryos and placentas.F8-/-Pros1-/- pregnant females were treated with low-dose aspirin (200mg/kg Lysine acetylsalicylate) or LMWH (Clexane, 100μg/mL delivered by osmotic pump 0.25 μL/hour) from E7 until E20 to assess if thromboprophylaxis could be beneficial for pregnancy.Results:No litters were produced from F8-/-Pros1-/- crosses.F8-/-Pros1-/- females were then mate with F8-/-Pros1+/- male and sacrificed at different gestational stages. A progressively high number of macerated and necrotic embryos were found after E9-E10 in comparison with pregnantcontrols: 0% (E9-10), 57% (E11-E12), 64% (E14-E15) and 67% (E16-E17). In parallel, number of embryos per gestation decreased in F8-/-Pros1-/- compared to F8-/-Pros1+/+ females (4±1 vs 9±1 at E11-E12, 3±1 vs 7±1 at E14-E15, 2±1 vs 7±2 at E16-E17, respectively). Macroscopically, the majority of alive embryos showed the presence of hemorrhages without PF. F8-/-Pros1-/- genotype was underrepresented among alive embryos (33% vs the expected 50%) and it disappeared from those collected after E12.5.The evaluation of DIC parameters in F8-/-Pros1-/- pregnant females during the period of experienced high embryonic mortality (E12-15) showed a significative reduction of platelet counts (486±105 vs 806±134 G/L F8-/-Pros1+/+, P<0.03), fibrinogen (1±0.1 vs 2±0.2 g/L, respectively, P<0.001) and increased TAT (33.2±5.1, 16.6±2.8 ng/L, respectively, P<0.02).Although these results suggest an activated state of blood coagulation, fibrin staining did not reveal fibrin deposition in lungs, liver and placenta from F8-/-Pros1-/- pregnant mice.Treatment of F8-/-Pros1-/- pregnant mice with LMWH or aspirin had a positive pregnancy outcome (n=4/female/treatment). However, litter size was reduced in comparison to F8-/-Pros1+/+ control mice. In particular, the aspirin group showed the lowest litter size while the LMWH group had increased pups number (3±1 vs 6±1, P<0.03). F8-/-Pros1-/- genotype was rescued only among newborns from LMWH treated group (n=1 over 7). Preliminary data from pups monitoring indicated that some pups were smaller, experienced hemorrhages and died after birth.Conclusion:Targeting F8 did not prevent pregnancy loss in F8-/-Pros1-/- mice. Only few F8-/-Pros1+/- embryos survived until E17 indicating that PS expression in the uteroplacental unit is indispensable for embryos development. F8-/-Pros1-/- pregnant mice displayed coagulation activation. However, further investigations are needed if the observed DIC is due to the lack of PS anticoagulant activity or to retained dead embryos. Aspirin or better LMWH partially prevented pregnancy loss indicating that thromboprophylaxis might be beneficial for pregnancy development with very low PS plasma levels. DisclosuresNo relevant conflicts of interest to declare.

Maternal Obesity Does Not Exacerbate the Effects of LPS Injection on Pregnancy Outcomes in Mice.

Simple SummaryObesity increases the risk of problems during pregnancy, potentially due to inappropriate activation of the immune system. We predicted that, because of this immune activation, obesity in mice would exacerbate the effects of lipopolysaccharide (LPS), a substance that mimics infection, impairs fetal growth and leads to pregnancy loss in animal models. Mice were fed a high- or low-fat diet for thirteen weeks prior to mating, and then received an LPS or control injection during pregnancy. Treatment with LPS induced pregnancy loss in some mice, as expected. However, LPS did not have more severe effects in females fed the high-fat diet, who were heavier. Our results therefore do not support the hypothesis that an otherwise healthy obese pregnancy can be driven to an adverse outcome by a low-level infection. Our study improves the understanding of why obese women are at greater risk of adverse pregnancy outcomes. In doing so, it will contribute to future studies that seek to determine how obese pregnancies at risk of adverse outcomes can be distinguished from healthy obese pregnancies.Obesity increases the risk of a number of pregnancy complications, potentially due to chronic inflammation. We predicted that an obesogenic high-fat diet (HFD) in mice would create an inflammatory environment that would exacerbate the effects of lipopolysaccharide (LPS), an inflammatory insult, administered during pregnancy. Females were placed on a HFD or a low-fat diet (LFD) prior to mating, injected with 2 µg LPS or control on gestational day 7 and collected on day 14. Treatment with LPS increased the odds that a female thought to be pregnant at injection had no conceptuses at day 14 (p = 0.024), suggesting that injection with LPS was more likely to induce complete abortion. However, there was no effect of diet on the odds of having no conceptuses at day 14 and no interaction between diet and LPS injection. Diet and LPS injection had no effect on the number of viable fetuses in females still pregnant at day 14. For fetal weight, there was a significant interaction between diet and treatment (p = 0.017), whereby LPS reduced fetal weight in HFD females but not in LFD females. However, LPS treatment of HFD females reduced fetal weight to that observed in control-injected LFD females. Although LPS increased the odds of abortion, there was little evidence that a HFD exacerbated the effects of LPS.

TGF-β1 Neutralization Improves Pregnancy Outcomes by Restoring Endometrial Receptivity in Mice with Adenomyosis.

The objective of this research is to study the effects of TGF-β1 inhibition on endometrial receptivity and pregnancy outcomes in mice with adenomyosis. Experiments were done using a mouse model of adenomyosis which took place in a hospital-affiliated laboratory. The mouse model used for this research is ICR mouse. Adenomyosis was induced by oral gavage of tamoxifen (TAM) from postnatal days (PNDs) 1 to 4 in ICR mice. Bilateral intrauterine injection of anti-TGF-β1-neutralizing antibody or isotype IgG or PBS was performed at PND42. The mice were then either sacrificed or mated at PND64 followed by sacrificing at gestational day (GD) 4 or proceeding to delivery. Implantation numbers, rate of dams with live birth, live birth numbers, survival at 1week old, and pup mortality rate after weaning were recorded. Collagen was demonstrated by Masson's trichrome and Van Gieson's stains. Uterine expression of a receptivity marker, leukemia inhibitory factor (LIF), was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC). Anti-TGF-β1 treatment increased the mean implantation numbers, fecundity rate, the rate of dams with live birth, pup survival rate at 1week old, and pup mortality rate after weaning. Collagen expression in uteri with adenomyosis was attenuated by anti-TGF-β1 treatment. Increased LIF expression by anti-TGF-β1 treatment was detected by qRT-PCR, Western blot, and IHC. The results suggest that inhibition of TGF-β1 improves pregnancy outcomes by restoring endometrial receptivity in mice with adenomyosis.

Effect of the IDO Gene on Pregnancy in Mice with Recurrent Pregnancy Loss.

The aim of this study is to investigate the effect of the IDO (indoleamine 2,3-dioxygenase) gene on pregnancy outcome in mice with recurrent pregnancy loss (RPL) and its mechanism of action in the maternal-fetal interface. An RPL model was established via natural mating of female CBA/J mice with male DBA/2 mice; thereafter, the female mice were randomly divided into groups treated with LV-EGFP (enhanced green fluorescent protein)-IDO (lentivirus vector carrying IDO-EGFP gene), LV-EGFP (negative control lentivirus vector), or phosphate-buffered saline (control). The mice were sacrificed at 13.5days of pregnancy, and the embryo absorption rate was determined. Peripheral blood regulatory T cells (Tregs) from the pregnant mice were detected using flow cytometry. Placental and decidual tissue IDO expression was detected using immunofluorescence and Western blotting. Inflammatory cell infiltration of the placental and decidual tissue was observed using hematoxylin-eosin (HE) staining. The LV-EGFP-IDO group had a significantly lower embryo absorption rate than the LV-EGFP and control groups (P = 0.0006 and P = 0.0049, respectively) and significantly more Tregs than the LV-EGFP and control groups (P = 0.0151 and P = 0.0392, respectively). Placental and decidual IDO protein levels correlated positively with peripheral blood Treg expression levels. The LV-EGFP-IDO group had significantly higher placental and decidual IDO protein levels than the LV-EGFP and control groups (P < 0.005), and it had significantly less inflammatory cell infiltration than the LV-EGFP and control groups. The IDO gene may reduce the embryo absorption rate in an RPL mouse model, possibly improving pregnancy outcome by upregulating Tregs and reducing the inflammatory response.

SAT-020 Fluoxetine Administration Influences Serotonin-Driven Bone Remodeling During Lactation and Pregnancy Outcome in Mice

Selective serotonin reuptake inhibitors (SSRI) are the most commonly prescribed class of antidepressants during pregnancy and lactation. SSRI decrease bone mineral density (BMD) across all ages and sexes. Lactation is also characterized by increased bone resorption to mobilize calcium due to the demands of lactation and achieves this via a serotonin-induced hormonal cascade. This serotonin-mediated bone loss is normally restored after weaning but is persistent when an SSRI is administered during the peripartum period. We hypothesize that the long-term BMD loss associated with fluoxetine administration during lactation will be seen in two sequential lactations and will have a more dramatic effect on maternal bone loss after the second lactation at both weaning and 3 months post-weaning. Female C57BL/6 mice were randomized to receive the SSRI fluoxetine hydrochloride (20 mg/kg) or saline daily from the beginning of pregnancy (E0), determined by the presence of a vaginal mucous plug, through the end of lactation (D21). They were then given a 3-week rest period before undergoing a second lactation with the same study design. Each group is paired to an age-matched virgin cohort to provide a non-lactating control. This created the following treatments: saline dam (S), fluoxetine dam (F), saline virgin pair (SV), and fluoxetine virgin pair (FV). Dual-energy X-ray absorptiometry (DEXA) was used to measure BMD of all groups throughout the peripartum period. At the end of gestation (E17.5), BMD was decreased in both the F and FV groups, and the BMD of the FV group remained persistently lower than the SV group across all timepoints. Both the F and S groups exhibited a dramatic decrease in BMD between E17.5 and the beginning of lactation (D2), then a slight increase between mid-lactation (D10) and at weaning (D21). There was an overall interaction between treatment and time for all groups across the peripartum period (p<0.0001). During the first peripartum period, the F dams that were mated had significantly increased pregnancy loss (p=0.0041) and pup mortality post-parturition compared to S mice. Of the dams that were inseminated, determined by the presence of a mucus plug, 63% of successfully mated S dams (n=19) resulted in a full-term pregnancy. Of those S dams (n=12), 25% lost their entire litter at the beginning of lactation, and 75% maintained their litter until weaning. In the F dams that were successfully mated (n=37), only 35% resulted in a full-term pregnancy. We conclude that fluoxetine administration during the peripartum period decreases long-term maternal BMD and that fluoxetine exhibits a drug-specific effect on the dam resulting in a notably decreased incidence of a successful pregnancy.

922: Effects of near-continuous low-dose neutron irradiation on pregnancy outcomes in mice

Now that orbital spaceflight is a continuous reality, deep space exploration and extra-Earth colonization have become the aspirational activities of space agencies and private entities. Understanding the impact of the extraterrestrial environment on human reproduction is a vital precursor to extra-Earth colonization, however, the effects of microgravity and cosmic radiation on reproductive physiology remain largely unknown. Relevant forms of cosmic radiation include protons, heavy ions, and the secondary neutron irradiation that occurs as protons bombard spacecrafts, habitats, or the human body, and these forms of radiation are fundamentally different than gamma or x-irradiation. We hypothesize that exposure of pregnant mice throughout gestation to chronic low-dose neutron irradiation would result in increased miscarriage and restricted fetal growth. Following copulatory plug positivity, eighty 9-13-week old female C57BL/6 mice were randomized to near-continuous neutron irradiation (21 hours/day) using Califonium-252 at a dose rate of 1 mGy/day vs. control (background: 0.005 mGy/day) for the duration of pregnancy (E0.5-E18.5) at NASA’s Colorado State University Neutron Radiation Facility. The dams were then randomized to euthanasia at either E12.5 to determine the rate of early miscarriage, or E18.5 to assess the rate of late miscarriage, fetal anomalies, and growth restriction. Placentas were weighed and stored for future secondary analyses including placental immunohistochemistry, cell signaling and nutrient transport, and gene expression studies. Phenotypic characteristics are provided in Table 1. Chronic low-dose neutron irradiation caused increased early resorption rate and significantly decreased placental weight. In contrast, there were no differences in birth length, birth weight, or anomaly rate between groups. Although this data cannot be directly extrapolated to humans, for the first time, we show that near-continuous low-dose neutron irradiation pan-pregnancy at a space-relevant dose rate leads to increased risk of miscarriage in a mouse model.

P10: Investigating the impact of herpes simplex virus type 2 infection on pregnancy outcomes in mice

American Journal of Reproductive ImmunologyVolume 81, Issue S1 p. 43-44 ABSTRACTS P10: Investigating the impact of herpes simplex virus type 2 infection on pregnancy outcomes in mice First published: 31 May 2019 https://doi.org/10.1111/aji.09_13120Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume81, IssueS1Special Issue: The 39th Annual Meeting of the American Society for Reproductive Immunology, 12-15 June 2019, Grand Rapids, Michigan, USAMay 2019Pages 43-44 RelatedInformation

The Deletion of TRPC6 Channels Perturbs Iron and Zinc Homeostasis and Pregnancy Outcome in Mice.

Transient receptor potential canonical 6 (TRPC6) protein is a nonselective cation channel permitting the uptake of essential elements such as iron (Fe) and zinc (Zn). TRPC6 is found throughout the body with high expression levels in the placenta. However, its role in this organ is still to be determined. To further advance our understanding of the physiological relevance of TRPC6, we have studied the placental histology, pregnancy outcome and the Fe and Zn status of organs (placenta, brain, kidney, liver and lung) collected from TRPC6 deficient (TRPC6-/-) mice and sex and age-matched C57Bl6/J and B6129SF2/J mice. Metal content was quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Quantitative reverse transcriptase PCR (qRT-PCR) and Western Blottings (WB) were performed to analyze the expression of placental markers and TRPC6. Our data show that TRPC6-/- mice displayed reduced litter sizes, structural changes of the placenta, along with altered mRNA levels of CD31 and Gcm1, two markers of placental development. Furthermore, immunoblots revealed elevated amounts of TRPC6 proteins in placentas from women diagnosed with preeclampsia, a common gestational disease. When compared to C57Bl6/J and B6129SF2/J, TRPC6-/- mice had elevated Zn levels in placenta, liver and kidney during embryonic development and postnatally, but not at adulthood. High amounts of Fe were found in the adult brain and liver of TRPC6-/- mice. The lung was however not affected by the deletion of TRPC6, indicating that this mouse strain developed organ and age-dependent perturbations in their Zn and Fe status. This work indicates that TRPC6 exerts critical pathophysiological functions in placenta, and provides further evidence for a role of this channel in the homeostasis of cations like Zn and Fe.

Trophoblast VIP deficiency entails immune homeostasis loss and adverse pregnancy outcome in mice.

Immune homeostasis maintenance throughout pregnancy is critical for normal fetal development. Trophoblast cells differentiate into an invasive phenotype and contribute to the transformation of maternal arteries and the functional shaping of decidual leukocyte populations. Insufficient trophoblast invasion, inadequate vascular remodeling, and a loss of immunologic homeostasis are associated with pregnancy complications, such as preeclampsia and intrauterine growth restriction. Vasoactive intestinal peptide (VIP) is a pleiotropic neuropeptide synthetized in trophoblasts at the maternal-placental interface. It regulates the function of trophoblast cells and their interaction with decidual leukocytes. By means of a murine model of pregnancy in normal maternal background with VIP-deficient trophoblast cells, here we demonstrate that trophoblast VIP is critical for trophoblast function: VIP gene haploinsufficiency results in lower matrix metalloproteinase 9 expression, and reduced migration and invasion capacities. A reduced number of regulatory T cells at the implantation sites along with a lower expression of proangiogenic and antiinflammatory markers were also observed. Findings detected in the implantation sites at early stages were followed by an abnormal placental structure and lower fetal weight. This effect was overcome by VIP treatment of the early pregnant mice. Our results support the relevance of trophoblast-synthesized VIP as a critical factor in vivo for trophoblast-cell function and immune homeostasis maintenance in mouse pregnancy.-Hauk, V., Vota, D., Gallino, L., Calo, G., Paparini, D., Merech, F., Ochoa, F., Zotta, E., Ramhorst, R., Waschek, J., Leirós, C. P. Trophoblast VIP deficiency entails immune homeostasis loss and adverse pregnancy outcome in mice.