Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110 nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

The effect of Gestational Diabetes Mellitus on the fetal compartment.

In indicated preterm births such a Gestational Diabetes Mellitus (GDM), little is known about the role of the amnion membranes. Investigating the role of amnion membrane inflammation in response GDM may suggest novel pathophysiologic mechanisms. We hypothesize that increased GDM inflammatory mediators may weaken the amnion membrane predisposing them to infection. Maternal and fetal serum and amnion membrane biopsies were collected from 20 GDM and 38 normoglycemic subjects (control) who underwent elective cesarean sections. Cytokines and adipokines were evaluated in serum and amnion culture supernatant samples. Amnion membrane biopsies from GDM and control subjects were studied: fresh frozen for RNA analysis for Toll-like receptor expression; cultured with LPS to test membrane permeability, and inflammation LPS + anti-TLR4 for testing mechanism. GDM was associated with higher fetal serum leptin (p = 0.004) and IL-10 (p = 0.04) compared to controls. Amnion membrane explants from GDM had higher levels of IL-6 (p = 0.019), and lower expression of Claudin-4 (p = 0.007) and increased permeability (p = 0.046) compared to controls. GDM membranes treated with LPS showed an increased expression of IL-10 (p = 0.013); IL-6 (p = 0.004) and TNF-α (p = 0.0005) but did not affect membrane permeability. LPS and anti-TLR4 antibody treatment reduced the production of TNF-α in controls (p = 0.03) and GDM (p = 0.007) compared to LPS alone. Fetal inflammatory response seems more balanced in GDM and does not impact membrane permeability function even with an infectious stimulus. Light fetal membrane inflammatory response may explain lack of preterm labor in GDM. Concluding, benign inflammation in the membranes may not be harmful for pregnancy maintenance.

Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release.

Preterm birth is a major contributor to neonatal mortality and morbidity. While the causes of preterm birth remain incompletely understood, infection is a major risk factor, and chorioamnionitis is commonly observed. Chorioamnionitis is characterized by inflammation and neutrophil infiltration of the fetal membranes (FM). We recently reported that human FMs which had been exposed to low levels of bacterial lipopolysaccharide (LPS) recruit neutrophils and activate them, increasing their secretion of pro-inflammatory cytokines, degranulation of myeloperoxidase (MPO), and release of neutrophil extracellular traps (NETs). Herein, we demonstrate that conditioned media (CM) from viral dsRNA (Poly(I:C))-stimulated FMs also increased neutrophil migration, and induced the secretion of inflammatory IL-8 and the release of NETs. Furthermore, CM from FMs stimulated by a combination of bacterial LPS and Poly(I:C) augmented neutrophil NET release, compared to CM from FMs stimulated with either Poly(I:C) or LPS alone. NETs induced by FMs exposed to Poly(I:C), with or without LPS, were released and degraded quicker than those induced by resting or LPS-stimulated FM-CM. These findings indicate that FMs exposed to viral dsRNA promote neutrophil recruitment, activation and NET formation, similar to FMs exposed to bacterial LPS alone. However, in response to FM polymicrobial stimulation the levels and kinetics of NET release are augmented. This work builds upon our understanding of how infections at the maternal-fetal interface may affect neutrophil function.

Activated Neutrophils Propagate Fetal Membrane Inflammation and Weakening through ERK and Neutrophil Extracellular Trap-Induced TLR-9 Signaling.

Preterm birth is associated with significant neonatal mortality and morbidity worldwide. Chorioamnionitis, inflammation of the fetal membranes (FMs), is a major risk factor and is characterized by neutrophil infiltration. However, the role of neutrophils at the FMs remains unclear. We recently reported that FMs exposed to bacterial LPS recruited more neutrophils compared with resting FMs and activated them to degranulate and release reactive oxygen species, chemokines/cytokines, and neutrophil extracellular traps. We posit that under resting conditions, neutrophils play a protective surveillance role, whereas during infection/inflammation, they induce FM tissue injury. To test this, human FM explants were exposed to neutrophil conditioned media (CM). We demonstrate that CM from neutrophils exposed to resting FM-CM did not affect FM viability or function. Conversely, CM from neutrophils activated by LPS-stimulated FM-CM significantly increased FM secretion of inflammatory IL-6, IL-8, GRO-α, and the markers of membrane weakening, MMP-9 and PGE2 This FM response was partially mediated by ERK signaling and neutrophil extracellular traps through the activation of the DNA sensor, TLR-9. Thus, neutrophils recruited by FMs during infection can propagate FM inflammation and weakening, acting in a feed-forward mechanism to propagate tissue injury at the maternal-fetal interface, increasing the risk of premature FM rupture and preterm birth in women with intrauterine infection.

Characterizing the immune cell population in the human fetal membrane.

PROBLEM: This study localized CD45+ immune cells and compared changes in their numbers between term, not in labor (TNIL) and term, labor (TL) human fetal membranes. METHOD OF STUDY: Fetal membranes (amniochorion) from normal TNIL and TL subjects were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry for evidence of total (CD45+ ) immune cells as well as innate immune cells (neutrophils, macrophages and NK cells) using specific markers. Fetal origin of immune cells was determined using polymerase chain reaction (PCR) for SRY gene in Y chromosome. RESULTS: CD45+ cells were localized in human fetal membranes for both TNIL and TL. A threefold increase in CD45+ cells was seen in TL fetal membranes of (7.73% ± 2.35) compared to TNIL (2.36% ± 0.78). This increase is primarily contributed by neutrophils. Macrophages and NK cells did not change in the membranes between TNIL and TL. Leukocytes of fetal origin are present in the fetal membranes. CONCLUSION: The fetal membranes without decidua contain a small proportion of immune cells. Some of these immune cells in the fetal membrane are fetal in origin. There is a moderate increase of immune cells in the fetal membranes at term labor; however, it is unclear whether this is a cause or consequence of labor. Further functional studies are needed to determine their contribution to membrane inflammation associated with parturition.

De novo Synthesis of SAA1 in the Placenta Participates in Parturition.

Serum amyloid A1 (SAA1) is an acute phase protein produced mainly by the liver to participate in immunomodulation in both sterile and non-sterile inflammation. However, non-hepatic tissues can also synthesize SAA1. It remains to be determined whether SAA1 synthesized locally in the placenta participates in parturition via eliciting inflammatory reactions. In this study, we investigated this issue by using human placenta and a mouse model. We found that SAA1 mRNA and protein were present in human placental villous trophoblasts, which was increased upon syncytialization as well as treatments with lipopolysaccharides (LPS), tumor necrosis factor-α (TNF-α), and cortisol. Moreover, significant increases in SAA1 abundance were observed in the placental tissue or in the maternal blood in spontaneous deliveries without infection at term and in preterm birth with histological chorioamnionitis. Serum amyloid A1 treatment significantly increased parturition-pertinent inflammatory gene expression including interleukin-1ß (IL-1ß), IL-8, TNF-α, and cyclooxygenase-2 (COX-2), along with increased PGF2α production in syncytiotrophoblasts. Mouse study showed that SAA1 was present in the placental junctional zone and yolk sac membrane, which was increased following intraperitoneal administration of LPS. Intraperitoneal injection of SAA1 not only induced preterm birth but also increased the abundance of IL-1ß, TNF-α, and COX-2 in the mouse placenta. Conclusively, SAA1 can be synthesized in the human placenta, which is increased upon trophoblast syncytialization. Parturition is accompanied with increased SAA1 abundance in the placenta. Serum amyloid A1 may participate in parturition in the presence and absence of infection by inducing the expression of inflammatory genes in the placenta.

Fetal membrane extracellular vesicle profiling reveals distinct pathways induced by infection and inflammation in vitro.

PROBLEM: Fetal inflammatory signals can be propagated to maternal tissues to initiate labor via exosomes (extracellular vesicles; 30-150 nm). We tested the hypothesis that fetal membrane cells exposed to infectious and inflammatory mediators associated with preterm birth (PTB) produce exosomes with distinct protein cargo contents indicative of underlying pathobiology. METHODS OF STUDY: Fetal membrane explants (FM) as well as primary amnion epithelial (AEC) and mesenchymal cells (AMC), and chorion cells (CC) from term deliveries were maintained in normal conditions (control) or exposed to LPS 100 ng/mL or TNF-α 50 ng/mL for 48 hours. Exosomes were isolated from media by differential centrifugation and size exclusion chromatography and characterized using cryo-electron microscopy (morphology), nanoparticle tracking analysis (size and quantity), Western blot (markers), and mass spectroscopy (cargo proteins). Ingenuity pathway analysis (IPA) determined pathways indicated by differentially expressed proteins. RESULTS: Irrespective of source or treatment, exosomes were spherical, had similar size, quantities, and markers (ALIX, CD63, and CD81). However, exosome cargo proteins were different between FM and individual fetal membrane cell-derived exosomes in response to treatments. Several common proteins were seen; however, there are several unique proteins expressed by exosomes from different cell types in response to distinct stimuli indicative of unique pathways and physiological functions in cells. CONCLUSIONS: We demonstrate collective tissue and independent cell response reflected in exosomes in response to infectious and inflammatory stimuli. These cargoes determined underlying physiology and their potential in enhancing inflammation in a paracrine fashion.

Relationship Between Pregnancy and Acute Disseminated Encephalomyelitis: A Single-Case Study.

The relationship between pregnancy and autoimmune diseases is unclear. This study investigated the possible role of local immune changes and the activation state of the HMGB1/TLR4/Nf-κB/IL-6 pathway at the maternal-fetal interface during pregnancy in the pathogenesis of acute disseminated encephalomyelitis (ADEM). Clinical data and blood samples of a patient with ADEM were collected to observe the dynamic changes in lymphocyte populations after an abortion. The expression of HMGB1, TLR4, Nf-κB, AQP4, IL-2, IL-4, IL-6, and TNF-α in the fetal membrane and placenta was compared between the patient with pregnancy-related ADEM and a woman with a normal pregnancy using Real-time qPCR and western blotting (WB). The patient was diagnosed with ADEM in the early stage of pregnancy after showing limb weakness symptoms. In the third month of gestation, the symptoms worsened, with a disturbance of consciousness and breathing. After the abortion, the patient relapsed with vertigo and visual rotation. Analysis of lymphocyte subsets by flow cytometry showed that B lymphocytes increased, while natural killer T lymphocytes decreased. WB and Real-time qPCR showed that the expression levels of HMGB1, TLR4, Nf-κB, AQP4, and IL-6 in the fetal membrane and placenta were higher in the patient with pregnancy-related ADEM than in the woman with a normal pregnancy, while those of IL-2 were lower in the patient than in the woman with a normal pregnancy. The local immune changes and the activation of the HMGB1/TLR4/Nf-κB/IL-6 pathway at the maternal-fetal interface may be related to the pathogenesis of ADEM.

Lipopolysaccharide-Stimulated Human Fetal Membranes Induce Neutrophil Activation and Release of Vital Neutrophil Extracellular Traps.

Preterm birth is a major contributor to neonatal mortality and morbidity, and infection is a major risk factor. Chorioamnionitis, inflammation of the placenta, and fetal membranes (FMs) are commonly observed in preterm birth and are characterized by neutrophil infiltration. However, interactions between FMs and neutrophils remain incompletely understood. The objectives of this study were to determine how FMs, with or without bacterial LPS stimulation, affect neutrophil recruitment, activation, and the formation of neutrophil extracellular traps (NETs) and to elucidate the signaling mechanisms involved. Using a combination of in vitro, ex vivo, and in vivo approaches, we show that human resting FMs can directly recruit neutrophils and induce them to produce proinflammatory factors. Furthermore, neutrophils release vital NETs in response to FM-derived factors. LPS-stimulated FMs further augmented neutrophil recruitment, inflammatory cytokine/chemokine secretion, and vital NET release and also induced reactive oxygen species production and degranulation. We demonstrate a role for FM-derived TNF-α in mediating these effects through activation of neutrophil p38 MAPK. We propose that, during infection, neutrophil recruitment and activation may neutralize pathogens, vital NET formation, and prolonged neutrophil viability, and in combination with degranulation, reactive oxygen species production and inflammatory chemokine/cytokine production may contribute to tissue injury at the maternal/fetal interface.

Expression and function of macrophage-inducible C-type lectin (Mincle) in inflammation driven parturition in fetal membranes and myometrium.

The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.

Fetal Membrane Inflammation Induces Preterm Birth Via Toll-Like Receptor 2 in Mice With Chronic Gingivitis.

Inflammation is associated with preterm birth. We previously described a mouse model of chronic inflammation-induced preterm birth after dental Porphyromonas gingivalis infection. The aim of this study was to employ this model system to investigate the mechanisms through which enhanced uterine contractility induces preterm birth. Messenger RNA (mRNA) encoding contraction-associated proteins, such as oxytocin receptors, was measured at various gestational time points by real-time polymerase chain reaction (PCR). Spontaneous and oxytocin-induced uterine contractile activity at gestational day 18 was assessed using a tissue organ bath. The expression levels of Toll-like receptor 2 (TLR2), TLR4, cyclooxygenase (COX)-2, nuclear factor-kappa B (NF-κB) p65, and p38 mitogen-activated protein kinase (MAPK) on gestational day 18 were also determined by real-time PCR or Western blotting. Messenger RNA encoding contraction-associated proteins was increased at gestational day 18, and the spontaneous contractile activity (1.6-fold greater area under the contraction curve) and sensitivity to oxytocin (EC50: 8.8 nM vs 2.2 nM) were enhanced in the P gingivalis group compared to those in the control group. In the P gingivalis group, COX-2 mRNA expression was not elevated in the placenta or myometrium but was upregulated 2.3-fold in the fetal membrane. The TLR2 mRNA levels in the fetal membrane were 2.7-fold higher in the P gingivalis group, whereas TLR4 levels were not elevated. Activation of the NF-κB p65 and p38 MAPK pathways was enhanced in the fetal membrane of the P gingivalis group. Thus, in mice with chronic dental P gingivalis infection, TLR2-induced inflammation in the fetal membrane leads to upregulation of uterine contractility, leading to preterm birth.

Abnormal lymphatic vessel development is associated with decreased decidual regulatory T cells in severe preeclampsia.

PROBLEM: The lymphatic vasculature controls leukocytes trafficking and limits the adaptive immune response. In previous models of preeclampsia (PE), defective immune function caused by disruption of lymphangiogenesis was shown to be involved in the disease pathophysiology. Especially, the dysfunction of regulatory T cells (Treg) at the maternal-fetal interface may be one of the causes of severe PE. In particular, activation of Tregs to obtain immune tolerance requires adequate antigen presentation through the lymphatic system. We hypothesized that impaired lymphangiogenesis and imbalanced Tregs at the maternal-fetal interface are associated with the pathophysiology of severe PE. However, the current research addressing this hypothesis is limited. Therefore, to compare differences in lymphangiogenesis in severe PE and normal conditions, we aimed to examine the location of lymphatics at the maternal-fetal interface and to investigate the association between lymphangiogenesis and Tregs in severe PE. METHOD OF STUDY: We obtained entire uterus from normal pregnant mice. Placental and fetal membranes, including decidua, were obtained from 10 pregnant women with severe PE and 10 gestational age-matched controls. Immunohistochemistry for LYVE1 was used to localize the distribution of lymphatic vessels and CD4, CD25, and FOXP3 for Treg. RESULTS: LYVE1-positive vessels were present in the uterine wall of mice. LYVE1-positive lymphatic vessels were localized on the human decidua. Tubular lymphatics were abundant in the control decidua, but significantly reduced in severe PE. Furthermore, lymphatic vessel density correlated with the number of decidual Tregs. CONCLUSION: Abnormal decidual lymphangiogenesis is associated with reduced numbers of decidual Tregs in severe PE.

Viral Infection Sensitizes Human Fetal Membranes to Bacterial Lipopolysaccharide by MERTK Inhibition and Inflammasome Activation.

Chorioamnionitis, premature rupture of fetal membranes (FMs), and subsequent preterm birth are associated with local infection and inflammation, particularly IL-1ß production. Although bacterial infections are commonly identified, other microorganisms may play a role in the pathogenesis. Because viral pandemics, such as influenza, Ebola, and Zika, are becoming more common, and pregnant women are at increased risk for associated complications, this study evaluated the impact that viral infection had on human FM innate immune responses. This study shows that a herpes viral infection of FMs sensitizes the tissue to low levels of bacterial LPS, giving rise to an exaggerated IL-1ß response. Using an ex vivo human FM explant system and an in vivo mouse model of pregnancy, we report that the mechanism by which this aggravated inflammation arises is through the inhibition of the TAM receptor, MERTK, and activation of the inflammasome. The TAM receptor ligand, growth arrest specific 6, re-establishes the normal FM response to LPS by restoring and augmenting TAM receptor and ligand expression, as well as by preventing the exacerbated IL-1ß processing and secretion. These findings indicate a novel mechanism by which viruses alter normal FM immune responses to bacteria, potentially giving rise to adverse pregnancy outcomes.

IL-27 induces a pro-inflammatory response in human fetal membranes mediating preterm birth.

Inflammation at the maternal-fetal interface has been shown to be involved in the pathogenesis of preterm birth. Interleukin 27 (IL-27), a heterodimeric cytokine, is known to mediate an inflammatory response in some pregnancy complications. In this study, we aimed to determine whether IL-27 could induce an inflammatory reaction at the maternal-fetal interface that would mediate the onset of preterm birth. We found elevated expression of IL-27 in human peripheral serum and elevated expression of its specific receptor (wsx-1) on fetal membranes in cases of preterm birth. Moreover, the release of inflammatory markers (CXCL10, IFN-γ, MCP-1, IL-6, IL-1ß and TNF-α), especially CXCL10, was markedly augmented upon stimulation of IL-27 in the fetal membranes. Additionally, IL-27 and IFN-γ cooperated to amplify the expression of CXCL10 in the fetal membranes. Moreover, the production of CXCL10 was increased in IL-27-treated fetal membrane through JNK, PI3K or Erk signaling pathways. Finally, MMP2 and MMP9 were activated by IL-27 in human fetal membranes, which may be related to the onset of preterm premature rupture of membranes (pPROM). In conclusion, for the first time, we reported that the aberrant expression of IL-27 could mediate an excessive inflammatory response in fetal membranes through the JNK, PI3K or Erk signaling pathways, which contributes to preterm birth.

Optineurin suppression activates the mediators involved in the terminal effector pathways of human labour and delivery.

Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.

Immune Modifications in Fetal Membranes Overlying the Cervix Precede Parturition in Humans.

In humans, parturition is currently viewed as an intrauterine outbreak of inflammation, accompanied by a massive release of proinflammatory cytokines at the maternal-fetal interface that comprises the maternal decidua, placenta, and fetal membranes. At term, fetal membranes overlying the cervix, the future site of rupture, show altered morphology and are termed the zone of altered morphology (ZAM). These alterations occur in normal fetal membranes during late pregnancy, in preparation for labor. In this study, transcriptome, flow cytometry, electron microscopy, and immunohistochemistry analyses collectively highlight a local shift in gene expression and lymphocyte activation in the ZAM. Just before labor, we show that highly polymorphic HLA-A, -B, and -C determinants of fetal origin are selectively exposed in the ZAM to the maternal immune system. A graft rejection-like program occurs in the ZAM, which involves 1) the activation of cytotoxic decidual NK cells, and 2) the decline of decidual immunotolerant M2-like macrophages. Comparison with a prior cohort of fetal membranes shows that acute inflammation only takes place after these first steps of immune modifications. Our results therefore strongly argue in favor of local immune remodeling at the onset of parturition.

Combinations and loads of bacteria affect the cytokine production by fetal membranes: An in vitro study.

PROBLEM: The polybacterial invasion and inflammation of the amniotic cavity is a common scenario in PTB, and then, we analyzed the cytokine production by human fetal membranes to better understand the host response to polybacterial infections. METHOD OF STUDY: Fetal membranes were treated by heat-inactivated genital mycoplasmas and Gardnerella vaginalis at 103 or 106 colony/color-forming units/mL alone or in combination. Cytokines/receptors were measured in the medium by immunoassays. RESULTS: Stimulation of genital mycoplasmas did not increase the proinflammatory cytokines, except Ureaplasma urealyticum that increased IL-8 levels. However, U. urealyticum and Mycoplasma hominis significantly increased IL-10 and IL-13 levels. G. vaginalis alone or in combination with genital mycoplasmas showed an increased proinflammatory and anti-inflammatory cytokines. CONCLUSIONS: G. vaginalis sustain a proinflammatory response in the fetal membranes in vitro, while genital mycoplasmas induce a strong control of the inflammatory response. The ability of genital mycoplasmas to control the proinflammatory response may favor their survival in the upper genital tract.

Cell-Free Fetal DNA, Telomeres, and the Spontaneous Onset of Parturition.

Multiple previous reports have provided compelling support for the premise that spontaneous parturition is mediated by activation of inflammation-related signaling pathways leading to increased secretion of cytokines and chemokines, the influx of neutrophils and macrophages into the pregnant uterus, increased production of uterine activation proteins (eg, connexin-43, cyclo-oxygenase-2, oxytocin receptors, etc), activation of matrix metalloproteinases, and the release of uterotonins leading to cervical ripening, membrane rupture, and myometrial contractions. The missing link has been the fetal/placental signal that triggers these proinflammatory events in the absence of microbial invasion and intrauterine infection. This article reviews the biomedical literature regarding the increase in cell-free fetal DNA (cffDNA), which is released during apoptosis in the placenta and fetal membranes at term, the ability of apoptosis modified vertebrate DNA to stimulate toll-like receptor-9 (TLR9) leading to increased release of cytokines and chemokines, and the potential "fail-safe" role for the anti-inflammatory cytokine IL-10. This article also reviews the literature supporting the key role that telomere loss plays in regard to increasing the ability of vertebrate (including placental) DNA to stimulate TLR9, and in regard to signaling the onset of apoptosis in the placenta and fetal membranes, thereby providing a biologic clock that determines the length of gestation and the timing for the onset of parturition. In summary, this literature review provides a strong rationale for future research to test the hypothesis that telomere loss and increased cffDNA levels trigger the proinflammatory events leading to the spontaneous onset of parturition in mammals: the "cffDNA/telomere hypothesis."

The role of placental MHC class I expression in immune-assisted separation of the fetal membranes in cattle.

The bovine fetus, like that of other species, is a semi-allograft and the regulation of materno-fetal alloimmunity is critical to prevent its immunological rejection. In cattle, a materno-fetal alloimmune response may be beneficial at parturition. It is hypothesized that upregulation of major histocompatibility complex (MHC) class I on the fetal membranes toward the end of gestation induces a maternal alloimmune response that activates innate immune effector mechanisms, aiding in the loss of the adherence between the fetal membranes and the uterus. Loss of fetal-maternal adherence is pivotal for the timely expulsion of the fetal membranes and the absence (or reduction) of the maternal immune response may lead to retained fetal membranes, a common reproductive disorder of cattle. Currently, there is no effective treatment for retained fetal membranes and a better understanding of materno-fetal alloimmune-assisted separation of the fetal membranes may lead to novel targets for the treatment of retained fetal membranes. In this review, the regulation of materno-fetal alloimmunity during pregnancy in cattle, with a focus on placental MHC class I expression, and the importance of maternal alloimmunity for the timely separation of the fetal membranes, are discussed.