Management of patients with endometriosis and infertility: laparoscopic treatment and spontaneous pregnancy rate.

OBJECTIVE: To evaluate factors involved in spontaneous pregnancy rate after surgery for endometriosis in patients with endometriosis and infertility. METHODS: This retrospective study spanned from 2014 to 2020 and included a follow-up period of two years of patients with endometriosis-related infertility who underwent laparoscopic surgery. Women aged 25 to 43 years with patent tubes, no/mild male factor and no other infertility factors were selected and grouped according to fertility management as follows: patients immediately prescribed ART (16.5%, ART-p); patients who chose not to undergo ART (83.5%) and achieved spontaneous pregnancy (71.8% SP-p); and patients who first chose not to undergo ART but had it subsequently (28.2%, NSP-p). RESULTS: A total of 200 patients were analyzed. Of the 167 patients who waited for spontaneous pregnancy, 71.8% achieved it. We observed a tendency of higher endometriosis ASRM scores in the ART-p group compared with patients who waited for spontaneous pregnancy, and lower scores in individuals that achieved spontaneous pregnancy. When we looked at how long it took to achieve pregnancy, we found that individuals in the SP-p group achieved pregnancy in 5.7 months, while subjects in the NSP-p group took 1.8 times longer than their peers in the SP-p group (p<0.001). However, once prescribed ART, the individuals in the NSP-p group achieved pregnancy within a similar time when compared with subjects in the SP-p group. In order to identify individuals that might benefit from ART early on, we performed a multivariable analysis and developed a decision tree (81.3% accuracy and 53.3% sensitivity). CONCLUSIONS: The present results indicated that, after surgery, the majority of patients achieved spontaneous pregnancy. The decision tree proposed in this study allows the early identification of patients who might require ART, thus decreasing the time between surgery and pregnancy and improving overall outcomes.

Monocyte immunometabolic reprogramming in human pregnancy: contribution of trophoblast cells.

Immunometabolism research is uncovering the relationship between metabolic features and immune cell functions in physiological and pathological conditions. Normal pregnancy entails a fine immune and metabolic regulation of the maternal-fetal interaction to assist the energetic demands of the fetus with immune homeostasis maintenance. Here, we determined the immunometabolic status of monocytes of pregnant women compared with nonpregnant controls and its impact on monocyte anti-inflammatory functions such as efferocytosis. Monocytes from pregnant women (16-20 wk) and nonpregnant age-matched controls were studied. Single cell-based metabolic assays using freshly isolated monocytes from both groups were carried out in parallel with functional assays ex vivo to evaluate monocyte efferocytic capacity. On the other hand, various in vitro metabolic assays with human monocytes or monocyte-derived macrophages were designed to explore the effect of trophoblast cells in the profiles observed. We found that pregnancy alters monocyte metabolism and function. An increased glucose dependency and enhanced efferocytosis were detected in monocytes from pregnant women at resting states, compared with nonpregnant controls. Furthermore, monocytes display a reduced glycolytic response when stimulated with lipopolysaccharide (LPS). The metabolic profiling of monocytes at this stage of pregnancy was comparable with the immunometabolic phenotypes of human monocytes treated in vitro with human first trimester trophoblast cell conditioned media. These findings suggest that immunometabolic mechanisms are involved in the functional shaping of monocytes during pregnancy with a contribution of trophoblast cells. Results provide new clues for future hypotheses regarding pregnancies complicated by metabolic disorders.NEW & NOTEWORTHY Immunometabolism stands as a novel perspective to understand the complex regulation of the immune response and to provide small molecule-based therapies. By applying this approach to study monocytes during pregnancy, we found that these cells have a unique activation pattern. They rely more on glycolysis and show increased efferocytosis/IL-10 production, but they do not have the typical proinflammatory responses. We also present evidence that trophoblast cells can shape monocytes into this distinct immunometabolic profile.

Extracellular Vesicles of Porphyromonas gingivalis Disrupt Trophoblast Cell Interaction with Vascular and Immune Cells in an In Vitro Model of Early Placentation.

Extracellular vesicles released by the primary pathogen of periodontal disease Porphyromonas gingivalis (Pg), referred to as outer membrane vesicles (OMVs), have been associated with the pathogenesis of systemic diseases like cardiovascular disease, rheumatoid arthritis, and Alzheimer's disease. A pathogenic role for Pg by disrupting placental homeostasis was proposed in the association between periodontal disease and adverse pregnancy outcomes. On the basis that trophoblast-derived factors modulate endothelial and immune cell profiles in normal pregnancy and the scarce presence of Pg in placenta, we hypothesized that OMVs from Pg affect trophoblast cell phenotype, impairing trophoblast-endothelium and trophoblast-neutrophil interactions. By means of in vitro designs with first-trimester human trophoblast cells, endothelial cells, and freshly isolated neutrophils, we showed that Pg OMVs are internalized by trophoblast cells and modulate the activity and expression of functional markers. Trophoblast cells primed with Pg OMVs enhanced neutrophil chemoattraction and lost their anti-inflammatory effect. In addition, reduced migration with enhanced adhesion of monocytes was found in endothelial cells upon incubation with the media from trophoblast cells pretreated with Pg OMVs. Taken together, the results support a pathogenic role of Pg OMVs at early stages of pregnancy and placentation through disruption of trophoblast contribution to vascular transformation and immune homeostasis maintenance.

Porphyromonas gingivalis outer membrane vesicles shape trophoblast cell metabolism impairing functions associated to adverse pregnancy outcome.

Periodontitis is proposed as a risk factor for preterm delivery, fetal growth restriction, and preeclampsia with severe consequences for maternal and neonatal health, but the biological mechanisms involved are elusive. Porphyromonas gingivalis gain access to the placental bed and impair trophoblast cell function, as assessed in murine and human pregnancy, suggesting a pathogenic role in adverse pregnancy and neonatal outcomes. P. gingivalis releases outer membrane vesicles (P. gingivalis OMV) during growth that spread to distant tissues and are internalized in host cells as described in metabolic, neurological, and vascular systemic diseases. Here we tested the hypothesis that P. gingivalis OMV internalized in trophoblast cells disrupt their metabolism leading to trophoblast and placenta dysfunction and adverse pregnancy outcomes. An in vitro design with human trophoblast cells incubated with P. gingivalis OMV was used together with ex vivo and in vivo approaches in pregnant mice treated with P. gingivalis OMV. P. gingivalis OMV modulated human trophoblast cell metabolism by reducing glycolytic pathways and decreasing total reactive oxygen species with sustained mitochondrial activity. Metabolic changes induced by P. gingivalis OMV did not compromise cell viability; instead, it turned trophoblast cells into a metabolic resting state where central functions such as migration and invasion were reduced. The effects of P. gingivalis OMV on human trophoblast cells were corroborated ex vivo in mouse whole placenta and in vivo in pregnant mice: P. gingivalis OMV reduced glycolytic pathways in the placenta and led to lower placental and fetal weight gain in vivo with reduced placental expression of the glucose transporter GLUT1. The present results point to OMV as a key component of P. gingivalis involved in adverse pregnancy outcomes, and even more, unveil a metabolic cue in the deleterious effect of P. gingivalis OMV on trophoblast cells and mouse pregnancy, providing new clues to understand pathogenic mechanisms in pregnancy complications and other systemic diseases.

miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization.

RESEARCH QUESTION: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? DESIGN: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. RESULTS: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1ß expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. CONCLUSIONS: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Targeting first trimester trophoblast cell metabolism modulates its susceptibility to Zika virus infection.

In the last 15 years Zika virus (ZIKV) caused several outbreaks of increasing scale in Micronesia, South Pacific islands, and more recently in the Caribbean and South America. The severity of the clinical presentation in neonates from pregnant women infected with ZIKV during the last outbreak supports the relevance of unraveling the mechanism of infection and viral persistence in the placenta with local viral isolates. Here, we investigated the relevance of trophoblast metabolic rewiring for viral multiplication and the role of the vasoactive intestinal peptide (VIP) as an endogenous factor associated with placental restriction to ZIKV infection at early pregnancy. Our in vitro model demonstrated that ZIKV triggers metabolic rewiring in first trimester cytotrophoblast-derived cells by increasing glucose utilization as fuel to sustain its replication, decreasing long-chain polyunsaturated fatty acid uptake, and promoting lipid droplets accumulation to favor its multiplication. Of note, variations in nutrient availability modulated viral spread in trophoblast cultures. The presence of VIP during trophoblast infection impaired ZIKV infective particle production and viral replication, restoring cell migration and metabolism. Moreover, the blockade of endogenous VIP signaling increased viral particle production and the viral entry receptor AXL expression. These results highlight the potential role of VIP as an endogenous antiviral factor related to trophoblast cell permissiveness to ZIKV infection at early pregnancy.

Vasoactive intestinal peptide deficiency promotes ovarian dysfunction associated to a proinflammatory microenvironment reminiscent of premature aging.

Complex immune regulation during pregnancy is required to ensure a successful pregnancy outcome. Vasoactive intestinal peptide (VIP) has local immunoregulatory effects on the ovary, uterus and maternal-fetal interface that favor a tolerogenic maternal microenvironment. Since the VIP Knockout (KO) mice are subfertile, we investigated the mechanisms underlying the effects of VIP deficiency on ovarian physiology and immune homeostasis. Therefore, we studied VIP KO, deficient (HT) and wild type (WT) female mice in estrus at 3 or 8 months of age. Young KO mice showed abnormal cycle timing and regularity associated with dysfunctional ovaries. Ovaries presented higher number of atretic follicles and reduced number of corpora lutea leading to a lower ovulation rates. Part of the VIP KO mice (25 %) failed to ovulate or ovulated oocytes incompetent to be fertilized (50 %). In particular, ovaries of young KO mice exhibited features of premature aging accompanied by a pro-inflammatory milieu with increased levels of IL-1ß. A unique macrophage subpopulation identified as "foamy macrophages" was found. On the other hand, aged VIP KO females did not gain body weight probably due to the sustained production of E2. Finally, the adoptive transfer of FOXP3+ cells to infertile VIP KO females resulted in their selective recruitment to the ovary. It increased FOXP3/RORγt and TGFß/IL-6 ratio improving ovarian microenvironment and pregnancy rate. The present results suggest that VIP contributes to ovarian homeostatic mechanisms required for a successful pregnancy.

VPAC1 and VPAC2 receptor deficiencies negatively influence pregnancy outcome through distinct and overlapping modulations of immune, trophoblast and vascular functions.

Pregnancy outcome relies on the maintenance of immune and metabolic homeostasis at the maternal fetal interface. Maternal and perinatal morbidity and mortality is associated with impaired placental development. Multiple regulatory effects of the endogenous-produced vasoactive intestinal peptide (VIP) on vascular, metabolic and immune functions at the maternal-fetal interface have been reported. Here we studied the involvement of the two primary high affinity receptors for VIP (VPAC1 and VPAC2) on maternal immune response, placental homeostasis and pregnancy outcome. Targeted disruption of each receptor gene led to altered placental structure, vascular and trophoblast functional markers and shaped the functional profiles of macrophages and neutrophils towards a proinflammatory state. Several changes in pregnant mice were receptor specific: ROS production elicited by VIP on neutrophils was selectively dependent on the presence of VPAC1 whereas apoptosis rate was associated with the VPAC2 deletion. In peritoneal macrophages from pregnant mice, levels of MHC-II, TLR2, and IL-10 were selectively altered in VPAC2 receptor-deficient mice, whereas IL-6 gene expression was reduced only in mice lacking VPAC1 receptors. Additionally, MMP9 mRNA in isolated TGCs was reduced in VPAC2 receptor deleted mice, while the percentage of IL-12 cells in post-phagocytosis macrophage cultures was selectively reduced in VPAC2 receptor deficient mice. The results indicate that manipulation of VPAC1 and VPAC2 receptor affects immune, vascular and metabolic environment at the maternal fetal interface. These mouse models offer new approaches to study pregnancy complications adding new perspectives to the development of VPAC receptor-selective drugs.

From decidualization to pregnancy progression: An overview of immune and metabolic effects of VIP.

BACKGROUND: A tight immune and metabolic regulation underlies the early maternal-placental interaction to assist the energetic dynamic demands of the fetus throughout pregnancy. The vasoactive intestinal peptide (VIP) holds biochemical, metabolic and immune properties consistent with a regulatory role during pregnancy. AIM: Here we overview critical aspects of embryo implantation and placental development with special focus on the immune and metabolic effects of VIP expressed by decidual and trophoblast cells. CONTENT: During decidualization, endometrial stromal cells undergo reticular stress and trigger unfolded protein response (UPR) that enable expansion of their endoplasmic reticulum and immunomodulatory factor synthesis. These processes appear differentially affected in recurrent abortion and in vitro fertilization failure suggesting their relevance in reproductive pathologies. Similarly, defective placentation associates with altered immune, vascular and trophoblast interaction resulting in complicated pregnancies that threaten maternal and neonatal health and underlie metabolic programming of adult life. We discuss the most recent research on decidual, trophoblast and immune cell interaction on the light of VIP regulation. Its role in decidualization and UPR associated with a sterile inflammatory response and angiogenesis is discussed. Evidence on VIP modulation of cytotrophoblast cell function, metabolism and immune profile is revised as well as the shaping of decidual leukocyte phenotype and function from decidualization to term. IMPLICATIONS: The broad spectrum of effects of VIP from implantation to term in normal and pathological conditions summarized here might contribute to the identification of novel biomarkers for diagnosis and pharmacological targeting.

Gingival crevicular fluid from pregnant women impairs trophoblast cell function and trophoblast-neutrophil interaction.

PROBLEM: A strong association between periodontitis and higher susceptibility to pregnancy complications like preeclampsia has been reported although the mechanisms remain elusive. Trophoblast cells modulate the recruitment and functional shaping of maternal leukocytes at early stages to sustain an antiinflammatory microenvironment and fetal growth. Neutrophil activation with reactive oxygen species (ROS) release is associated with preeclampsia. Our aim was to study the effect of the gingival crevicular fluid (GCF) from pregnant women on trophoblast cell function and trophoblast-neutrophil interaction. METHOD OF STUDY: Pregnant women at 16-20 weeks of gestation (n = 27) and non-pregnant women (n = 8) as the control group were studied for gingivoperiodontal clinical score evaluation and GCF collection. Total bacteria and common periodontal pathogens were analyzed in GCF samples. The effect of each GCF sample was tested on first trimester trophoblast-derived cells to assess cell migration, cytokine expression and glucose uptake. Also, the effect of GCF on human peripheral neutrophil chemoattraction by trophoblast cells and ROS formation was assessed. RESULTS: Gingival crevicular fluid from pregnant women reduced trophoblast cell migration, increased proinflammatory marker expression and glucose uptake. A significant correlation between gingivoperiodontal score and trophoblast dysfunction was observed. Upon conditioning of trophoblast cells with GCF, only the GCF from pregnant women stimulated neutrophil chemoattraction. Similarly, GCF from pregnant but not from non-pregnant controls stimulated ROS formation in neutrophils. CONCLUSIONS: Gingival crevicular fluid from pregnant women is deleterious for first trimester trophoblast cell function. These effects could lead to placental homeostasis disruption underlying a pathogenic mechanism of pregnancy complications associated to periodontal disease.

Maternal selection of human embryos in early gestation: Insights from recurrent miscarriage.

Compared to most mammals, human pregnancy is unusual in that it involves chromosomally diverse embryos, cyclical breakdown and regeneration of the uterine mucosa, and intimate integration of fetal and maternal cells at the uteroplacental interface. Not surprisingly, pregnancy often falters in early gestation. Whether these losses result in clinical miscarriages depends on the origins and impacts of chromosomal errors on fetal development and the ability of the decidualizing endometrium to engage in embryo biosensing and selection. Aneuploidy originating in oocytes during meiosis drives the age-related risk of miscarriage. By contrast, the frequency of endometrial cycles with an impaired decidual response may account for the stepwise increase in miscarriage rates with each pregnancy loss independently of maternal age. Additional physiological mechanisms operate in early gestation to ensure that most failing pregnancies are lost before vascular maternal-fetal connections are established by the end of the first trimester. Here, we summarise how investigations into the mechanisms that cause miscarriage led to new insights into the processes that govern maternal selection of human embryos in early gestation.

Understanding the natural selection of human embryos: Blastocyst quality modulates the inflammatory response during the peri-implantation period.

PROBLEM: Decidualized cells display an active role during embryo implantation sensing blastocyst quality, allowing the implantation of normal developed blastocysts and preventing the invasion of impaired developed ones. Here, we characterized the immune microenvironment generated by decidualized cells in response to soluble factors secreted by blastocysts that shape the receptive milieu. METHOD OF STUDY: We used an in vitro model of decidualization based on the Human Endometrial Stromal Cells line (HESC) differentiated with medroxiprogesterone and dibutyryl-cAMP, then treated with human blastocysts-conditioned media (BCM) classified according to their quality. RESULTS: Decidualized cells treated with BCM from impaired developed blastocysts increased IL-1ß production. Next, we evaluated the ability of decidualized cells to modulate other mediators associated with menstruation as chemokines. Decidualized cells responded to stimulation with BCM from impaired developed blastocysts increasing CXCL12 expression and CXCL8 secretion. The modulation of these markers was associated with the recruitment and activation of neutrophils, while regulatory T cells recruitment was restrained. These changes were not observed in the presence of BCM from normal developed blastocysts. CONCLUSION: Soluble factors released by impaired developed blastocysts induce an exacerbated inflammatory response associated with neutrophils recruitment and activation, providing new clues to understand the molecular basis of the embryo-endometrial dialogue.

Growth impairment, increased placental glucose uptake and altered transplacental transport in VIP deficient pregnancies: Maternal vs. placental contributions.

Glucose uptake by the placenta and its transfer to the fetus is a finely regulated process required for placental and fetal development. Deficient placentation is associated with pregnancy complications such as fetal growth restriction (FGR). The vasoactive intestinal peptide (VIP) has embryotrophic effects in mice and regulates human cytotrophoblast metabolism and function. Here we compared glucose uptake and transplacental transport in vivo by VIP-deficient placentas from normal or VIP-deficient maternal background. The role of endogenous VIP in placental glucose and amino acid uptake was also investigated. Wild type C57BL/6 (WT) or VIP+/- (VIP HT) females were mated with WT, VIP knock-out (VIP KO) or VIP HT males. Glucose uptake and transplacental transport were evaluated by the injection of the fluorescent d-glucose analogue 2-NBDG in pregnant mice at gestational day (gd) 17.5. Glucose and amino acid uptake in vitro by placental explants were measured with 2-NBDG or 14C-MeAIB respectively. In normal VIP maternal background, fetal weight was reduced in association with placental VIP deficiency, whereas placental weight was unaltered. Paradoxically, VIP+/- placentas presented higher glucose uptake and higher gene expression of GLUT1 and mTOR than VIP+/+ placentas. However, in a maternal VIP-deficient environment placental uptake and transplacental transport of glucose increased while fetal weights were unaffected, regardless of feto-placental genotype. Results point to VIP-deficient pregnancy in a normal background as a suitable FGR model with increased placental glucose uptake and transplacental transport. The apparently compensatory actions are unable to sustain normal fetal growth and could result in complications later in life.

In Vitro Identification and Isolation of Human Neutrophil Extracellular Traps.

Neutrophils release web like-structures known as neutrophil extracellular traps (NETs) that ensnare and kill microorganisms. These networks are constituted of a DNA scaffold with associated antimicrobial proteins, which are released to the extracellular space as an effective mechanism to fight against invading microorganisms. In parallel with this beneficial role to avoid microbial dissemination and wall off infections, accumulating evidence supports that under certain circumstances, NETs can exert deleterious effects in inflammatory, autoimmune, and thrombotic pathologies. Research on NET properties and their role in pathophysiological processes is a rapidly evolving and expanding field. Here, we describe a combination of methods to achieve a successful in vitro NET visualization, semiquantification, and isolation.

Miscarriage matters: the epidemiological, physical, psychological, and economic costs of early pregnancy loss.

Miscarriage is generally defined as the loss of a pregnancy before viability. An estimated 23 million miscarriages occur every year worldwide, translating to 44 pregnancy losses each minute. The pooled risk of miscarriage is 15·3% (95% CI 12·5-18·7%) of all recognised pregnancies. The population prevalence of women who have had one miscarriage is 10·8% (10·3-11·4%), two miscarriages is 1·9% (1·8-2·1%), and three or more miscarriages is 0·7% (0·5-0·8%). Risk factors for miscarriage include very young or older female age (younger than 20 years and older than 35 years), older male age (older than 40 years), very low or very high body-mass index, Black ethnicity, previous miscarriages, smoking, alcohol, stress, working night shifts, air pollution, and exposure to pesticides. The consequences of miscarriage are both physical, such as bleeding or infection, and psychological. Psychological consequences include increases in the risk of anxiety, depression, post-traumatic stress disorder, and suicide. Miscarriage, and especially recurrent miscarriage, is also a sentinel risk marker for obstetric complications, including preterm birth, fetal growth restriction, placental abruption, and stillbirth in future pregnancies, and a predictor of longer-term health problems, such as cardiovascular disease and venous thromboembolism. The costs of miscarriage affect individuals, health-care systems, and society. The short-term national economic cost of miscarriage is estimated to be £471 million per year in the UK. As recurrent miscarriage is a sentinel marker for various obstetric risks in future pregnancies, women should receive care in preconception and obstetric clinics specialising in patients at high risk. As psychological morbidity is common after pregnancy loss, effective screening instruments and treatment options for mental health consequences of miscarriage need to be available. We recommend that miscarriage data are gathered and reported to facilitate comparison of rates among countries, to accelerate research, and to improve patient care and policy development.

Decidual factors and vasoactive intestinal peptide guide monocytes to higher migration, efferocytosis and wound healing in term human pregnancy.

AIM: To explore the functional profile of circulating monocytes and decidual macrophages at term human pregnancy and their contribution to tissue repair upon stimulation ex vivo with decidual factors and the vasoactive intestinal peptide (VIP). METHODS: Peripheral blood monocytes were isolated from pregnant and non-pregnant volunteers and tested in vitro with decidual explants from term placenta and VIP. The effect of VIP on decidual explants and the effect of its conditioned media on monocytes or decidual macrophages isolated by magnetic beads was carried out by RT-qPCR and ELISA for cytokines expression and release. Migration assays were performed in transwell systems. Efferocytosis was assessed in monocytes or decidual macrophages with CFSE-labelled autologous apoptotic neutrophils and quantified by flow cytometry. Monocyte and decidual macrophages wound healing capacity was evaluated using human endometrial stromal cell monolayers. Immunohistochemistry was performed in serial tissue sections of different placentas. RESULTS: VIP is expressed in the villi as well as in trophoblast giant cells distributed within the decidua of term placenta. VIP induced the expression of antiinflmammatory markers and monocyte chemoattractant CCL2 and CCL3 in decidual tissues. Monocytes presented higher migration towards decidual explants than CD4 and CD8 cells. VIP-conditioned monocytes displayed an enhanced efferocytosis and wound healing capacity comparable to that of decidual macrophages. Moreover limited efferocytosis of pregnant women monocytes was restored by VIP-induced decidual factors. CONCLUSION: Results show the conditioning of monocytes by decidual factors and VIP to sustain processes required for tissue repair and homeostasis maintenance in term placenta.

Zika virus infection of first trimester trophoblast cells affects cell migration, metabolism and immune homeostasis control.

Zika virus (ZIKV) re-emerged after circulating almost undetected for many years and the last spread in 2015 was the major outbreak reported. ZIKV infection was associated with congenital fetal growth anomalies such as microcephaly, brain calcifications, and low birth weight related to fetal growth restriction. In this study, we investigated the effect of ZIKV infection on first trimester trophoblast cell function and metabolism. We also studied the interaction of trophoblast cells with decidual immune populations. Results presented here demonstrate that ZIKV infection triggered a strong antiviral response in first trimester cytotrophoblast-derived cells, impaired cell migration, increased glucose uptake and GLUT3 expression, and reduced brain derived neurotrophic factor (BDNF) expression. ZIKV infection also conditioned trophoblast cells to favor a tolerogenic response since an increased recruitment of CD14+ monocytes bearing an anti-inflammatory profile, increased CD4+ T cells and NK CD56Dim and NK CD56Bright populations and an increment in the population CD4+ FOXP3+ IL-10+ cells was observed. Interestingly, when ZIKV infection of trophoblast cells occurred in the presence of the vasoactive intestinal peptide (VIP) there was lower detection of viral RNA and reduced toll-like receptor-3 and viperin messenger RNA expression, along with reduced CD56Dim cells trafficking to trophoblast conditioned media. The effects of ZIKV infection on trophoblast cell function and immune-trophoblast interaction shown here could contribute to defective placentation and ZIKV persistence at the fetal-maternal interface. The inhibitory effect of VIP on ZIKV infection of trophoblast cells highlights its potential as a candidate molecule to interfere ZIKV infection during early pregnancy.

Decidualization Process Induces Maternal Monocytes to Tolerogenic IL-10-Producing Dendritic Cells (DC-10).

Decidualization is a process that involves phenotypic and functional changes of endometrial stromal cells to sustain endometrial receptivity and the participation of immunoregulatory factors to maintain immune homeostasis. In this context, tolerogenic dendritic cells (DCs) can induce regulatory T cells, which are essential to manage the pro- to anti-inflammatory transition during embryo implantation. Recently, Myeloid Regulatory Cells (MRCs) were proposed as immunosuppressants and tolerance-inducer cells, including the DC-10 subset. This novel and distinctive subset has the ability to produce IL-10 and to induce type 1 regulatory T cells (Tr1) through an HLA-G pathway. Here we focus on the impact of the decidualization process in conditioning peripheral monocytes to MRCs and the DC-10 subset, and their ability to induce regulatory T cells. An in vitro model of decidualization with the human endometrial stromal cell line (HESC), decidualized by medroxyprogesterone and dibutyryl-cAMP was used. Monocytes isolated from peripheral blood mononuclear cells from healthy women were cultured with rhGM-CSF + rhIL-4 and then, the effect of conditioned media from decidualized (Dec-CM) and non-decidualized cells (Non-dec-CM) was tested on monocyte cultures. We found that Dec-CM inhibited the differentiation to the CD1a+CD14- immature DC profile in a concentration-dependent manner. Dec-CM also significantly increased the frequency of CD83+CD86low and HLA-DR+ cells in the monocyte-derived culture. These markers, associated with the increased production of IL-10, are consistent with a MRCs tolerogenic profile. Interestingly, Dec-CM treatment displayed a higher expression of the characteristic markers of the tolerogenic DC-10 subset, HLA-G and ILT2/CD85j; while this modulation was not observed in cultures treated with Non-dec-CM. Moreover, when monocyte cultures with Dec-CM were challenged with LPS, they sustained a higher IL-10 production and prevented the increase of CD83, CD86, IL-12p70, and TNF-α expression. Finally, the DC-10 subset was able to induce a CD4+HLA-G+ regulatory T cells subset. These results suggest that the decidualization process might induce different subsets of MRCs, like DC-10, able to induce regulatory T cells as a novel CD4+HLA-G+ subset which might play an immunoregulatory role in embryo implantation.

Human Chorionic Gonadotropin modulates CXCL10 Expression through Histone Methylation in human decidua.

The process of implantation, trophoblast invasion and placentation demand continuous adaptation and modifications between the trophoblast (embryonic) and the decidua (maternal). Within the decidua, the maternal immune system undergoes continued changes, as the pregnancy progress, in terms of the cell population, phenotype and production of immune factors, cytokines and chemokines. Human chorionic gonadotropin (hCG) is one of the earliest hormones produced by the blastocyst and has potent immune modulatory effects, especially in relation to T cells. We hypothesized that trophoblast-derived hCG modulates the immune population present at the maternal fetal interface by modifying the cytokine profile produced by the stromal/decidual cells. Using in vitro models from decidual samples we demonstrate that hCG inhibits CXCL10 expression by inducing H3K27me3 histone methylation, which binds to Region 4 of the CXCL10 promoter, thereby suppressing its expression. hCG-induced histone methylation is mediated through EZH2, a functional member of the PRC2 complex. Regulation of CXCL10 expression has a major impact on the capacity of endometrial stromal cells to recruit CD8 cells. We demonstrate the existence of a cross talk between the placenta (hCG) and the decidua (CXCL10) in the control of immune cell recruitment. Alterations in this immune regulatory function, such as during infection, will have detrimental effects on the success of the pregnancy.

Immunoregulation of the decidualization program: focus on the endoplasmic reticulum stress.

Decidualization denotes the reprogramming of endometrial stromal cells that includes the secretion of different mediators like cytokines, chemokines, and the selective recruitment of immune cells. This physiological process involves changes in the secretome of the endometrial stromal cells leading to the production of immunomodulatory factors. The increased amount of protein secretion is associated with a physiological endoplasmic reticulum (ER) stress and the resulting unfolded protein response (UPR), allowing the expansion of ER and the machinery to assist the protein folding. Notably, the signaling pathways involved in the ER stress and the UPR are interconnected with the onset of a sterile inflammatory response, as well as with angiogenesis. Both of these processes have a key role in decidualization and placentation, therefore, alterations in them could lead to pregnancy complications. In this review, we will discuss how the induction of ER stress and the UPR processes that accompanies the decidualization are associated with embryo implantation and whether they might condition pregnancy outcome. The ER stress activates/triggers sensing proteins which, among others, induces kinase/RNAse-TXNIP expression, activating the NLRP3 inflammasome. This multiprotein system allows caspase-1 activation, which catalyzes the cleavage of the inactive IL-1ß proform toward the mature secretory form, with pro-implantatory effects. However, the sterile inflammatory response should be later controlled in favor of a tolerogenic microenvironment to sustain pregnancy. In accordance, alterations of the ER stress and UPR processes can be reflected in recurrent implantation failures (RIF), recurrent pregnancy loss (RPL), or complications associated with deficient placentation, such as preeclampsia (PE).