Fetal Trophoblast Research Articles

OP049: Automated isolation and genetic characterization of single circulating fetal trophoblasts in pregnant women

In the past decades, the existence of fetal cells circulating in the maternal blood during the first trimester of pregnancy has been extensively proved. The isolation as single intact cells and the subsequent genetic characterization of these fetal extravillous trophoblasts (fEVTs) could represent a valuable resource for prenatal diagnosis. Given the rarity of fetal cells in the maternal circulation and technical challenges related to their enrichment, a non-invasive isolation from maternal blood using an automated approach is still an unmet clinical need.

CD39/CD73 Dysregulation of Adenosine Metabolism Increases Decidual Natural Killer Cell Cytotoxicity: Implications in Unexplained Recurrent Spontaneous Abortion.

Unexplained recurrent spontaneous abortion (URSA) is believed to be associated with impaired immunosuppression at the maternal-fetal interface, but the detailed molecular mechanism remains unclear. The ATP-adenosine metabolic pathway regulated by CD39/CD73 has recently been recognized to be important in immunosuppression. This study aimed to investigate the regulation of decidual natural killer (dNK) cells and fetal extravillous trophoblast (EVT) cells by CD39 and CD73 in URSA, as well as the possible regulatory mechanism of CD39/CD73 via the TGF-β-mTOR-HIF-1α pathway using clinical samples and cell models. Fewer CD39+ and CD73+ cells were found in the URSA decidual and villous tissue, respectively. Inhibition of CD39 on dNK cells transformed the cells to an activated state with increased toxicity and decreased apoptosis, and changed their cytokine secretion, leading to impaired invasion and proliferation of the co-cultured HTR8/SVneo cells. Similarly, inhibition of CD73 on HTR8/SVneo cells decreased the adenosine concentration in the cell culture media, increased the proportion of CD107a+ dNK cells, and decreased the invasion and proliferation capabilities of the HTR8/SVneo cells. In addition, transforming growth factor-β (TGF-β) triggered phosphorylation of mammalian target of rapamycin (mTOR) and Smad2/Smad3, which subsequently activated hypoxia-inducible factor-1α (HIF-1α) to induce the CD73 expression on the HTR8/SVneo cells. In summary, reduced numbers of CD39+ and CD73+ cells at the maternal-fetal interface, which may be due to downregulated TGF-β-mTOR-HIF-1α pathway, results in reduced ATP-adenosine metabolism and increased dNK cytotoxicity, and potentially contributes to URSA occurrences.

Decidual NK cells kill Zika virus–infected trophoblasts

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal-fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 -/- pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.

OC05.01: Automated isolation of single circulating fetal trophoblasts for prenatal genetic analysis

OC05.01: Automated isolation of single circulating fetal trophoblasts for prenatal genetic analysis

Inhibition of the C-X-C Motif Chemokine 12 (CXCL12) and Its Receptor CXCR4 Reduces Utero-Placental Expression of the VEGF System and Increases Utero-Placental Autophagy.

The placenta, a unique organ that only develops during pregnancy, is essential for nutrient, oxygen, and waste exchange between offspring and mother. Yet, despite its importance, the placenta remains one of the least understood organs and knowledge of early placental formation is particularly limited. Abnormalities in placental development result in placental dysfunction or insufficiency whereby normal placental physiology is impaired. Placental dysfunction is a frequent source of pregnancy loss in livestock, inflicting serious economic impact to producers. Though the underlying causes of placental dysfunction are not well-characterized, initiation of disease is thought to occur during establishment of functional fetal and placental circulation. A comprehensive understanding of the mechanisms controlling placental growth and vascularization is necessary to improve reproductive success in livestock. We propose chemokine C-X-C motif ligand 12 (CXCL12) signaling through its receptor CXCR4 functions as a chief coordinator of vascularization through direct actions on fetal trophoblast and maternal endometrial and immune cells. To investigate CXCL12–CXCR4 signaling on uteroplacental vascular remodeling at the fetal–maternal interface, we utilized a CXCR4 antagonist (AMD3100). On day 12 post-breeding in sheep, osmotic pumps were surgically installed and delivered either AMD3100 or saline into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 35 of ovine pregnancy, fetal/placental and endometrial tissues were collected, snap-frozen in liquid nitrogen, and uterine horn cross sections were preserved for immunofluorescent analysis. Suppressing CXCL12–CXCR4 at the fetal–maternal interface during initial placental vascularization resulted in diminished abundance of select angiogenic factors in fetal and maternal placenta on day 35. Compared to control, less vascular endothelial growth factor (VEGF) and VEFG receptor 2 (KDR) were observed in endometrium when CXCL12–CXCR4 was diminished. Less VEGF was also evident in fetal placenta (cotyledons) in ewes receiving AMD3100 infusion compared to control. Suppressing CXCL12–CXCR4 at the fetal–maternal interface also resulted in greater autophagy induction in fetal and maternal placenta compared to control, suggestive of CXCL12–CXCR4 impacting cell survival. CXCL12–CXCR4 signaling may govern placental homeostasis by serving as a critical upstream mediator of vascularization and cell viability, thereby ensuring appropriate placental development.

Differential Expression Pattern of Retroviral Envelope Gene in the Equine Placenta.

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal–maternal relationship. To this end, we used reverse transcription–quantitative PCR (RT–qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus–mother relationship can be hypothesized.

Polymorphisms in LILRB-1 affects its ligand interactions

Abstract Leukocyte immunoglobulin-like receptor B-1 (LILRB-1) is an inhibitory receptor of the immunoglobulin super family expressed on a subset of natural killer (NK) cells. LILRB-1 binds a wide range of classical and non-classical MHC class I molecules, and a glycoprotein product of the human cytomegalovirus (HCMV), UL18. UL18 is a structural homolog of MHC class I expressed on the surface of infected cells. The two most distal domains of LILRB-1, denoted as D1 and D2, are the main contributors at the interaction interface of LILRB-1 with its ligands. Four non-synonymous polymorphisms within D1 and D2 domains produce LILRB-1’s natural variants. Interestingly, these variants have shown different expression and binding trends on NK cells and were suggested to affect susceptibility to HCMV infection in a cohort of kidney transplant recipients. Differences in the interaction of different LILRB-1 variants with their ligands such as, classical (HLA-Cw15) and non-classical MHC class I ligands (HLA-G) as well as UL18 have are assessed using a flowcytometry based binding assay and confirmed with functional assays. Our experiments show a trend of tight binding range of most LILRB-1 variants with HLA-G. However, the strength of binding is more variant with other classical MHC class I ligands, showing a constant trend based on the protein introduced by each of the four non-synonymous polymorphisms in different genotypes. Such findings may highlight an evolutionary pressure to maintain a relatively constant interaction of LILRB-1 with HLA-G, which is expressed on fetal trophoblasts to maintain maternal tolerance.

Insights Into Extracellular Vesicle/Exosome and miRNA Mediated Bi-Directional Communication During Porcine Pregnancy.

Spontaneous fetal loss is one of the most important challenges that commercial pig industry is still facing in North America. Research over the decade provided significant insights into some of the associated mechanisms including uterine capacity, placental efficiency, deficits in vasculature, and immune-inflammatory alterations at the maternal-fetal interface. Pigs have unique epitheliochorial placentation where maternal and fetal layers lay in opposition without any invasion. This has provided researchers opportunities to accurately tease out some of the mechanisms associated with maternal-fetal interface adaptations to the constantly evolving needs of a developing conceptus. Another unique feature of porcine pregnancy is the conceptus derived recruitment of immune cells during the window of conceptus attachment. These immune cells in turn participate in pregnancy associated vascular changes and contribute toward tolerance to the semi-allogeneic fetus. However, the precise mechanism of how maternal-fetal cells communicate during the critical times in gestation is not fully understood. Recently, it has been established that bi-directional communication between fetal trophoblasts and maternal cells/tissues is mediated by extracellular vesicles (EVs) including exosomes. These EVs are detected in a variety of tissues and body fluids and their role has been described in modulating several physiological and pathological processes including vascularization, immune-modulation, and homeostasis. Recent literature also suggests that these EVs (exosomes) carry cargo (nucleic acids, protein, and lipids) as unique signatures associated with some of the pregnancy associated pathologies. In this review, we provide overview of important mechanisms in porcine pregnancy success and failure and summarize current knowledge about the unique cargo containing biomolecules in EVs. We also discuss how EVs (including exosomes) transfer their contents into other cells and regulate important biological pathways critical for pregnancy success.

SARS-CoV-2 colonization of maternal and fetal cells of the human placenta promotes alteration of local renin-angiotensin system

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection appears to increase the risk of adverse pregnancy outcomes, such as pre-eclampsia in pregnant women. The mechanism(s) by which this occurs remains unclear.MethodsWe investigated the pathophysiology of SARS-CoV-2 at maternal-fetal interface in pregnant women who tested positive for the virus using RNA in situ hybridization (viral RNA), immunohistochemistry, and hematoxylin and eosin staining. To investigate whether viral infection alters the renin angiotensin system (RAS) in placenta, which controls blood pressure, we treated human trophoblasts with recombinant spike protein or a live modified virus with a vesicular stomatitis viral backbone expressing spike protein (VSV-S).FindingsViral colonization was highest in maternal decidua, fetal trophoblasts, Hofbauer cells, and in placentas delivered prematurely. We localized SARS-CoV-2 to cells expressing angiotensin-converting enzyme 2 (ACE2) and demonstrate that infected placentas had significantly reduced ACE2. In response to both spike protein and VSV-S, cellular ACE2 decreased although angiotensin II receptor type 1 (AT1R) increased with concomitant increase in soluble fms-like tyrosine kinase-1 (sFlt1). Viral infection decreased pro-angiogenic factors, AT2R, and placental growth factor, which competitively binds to sFlt1. Sera from infected pregnant women had elevated levels of sFlt1 and angiotensin II type 1-receptor autoantibodies prior to delivery, both signatory markers of pre-eclampsia.ConclusionsSARS-CoV-2 colonizes ACE2-expressing maternal and fetal cells in the placenta. Infection in pregnant women correlates with alteration of placental RAS. As RAS regulates blood pressure, SARS-CoV-2 infection may thus increase adverse hemodynamic outcomes, such as pre-eclampsia in pregnant women.FundingNIH/NICHD grants R01 HD091218 and 3R01HD091218-04S1 (RADx-UP Supplement).

Do uterine PTGS2, PGFS, and PTGFR expression play a role in canine uterine inertia?

The aetiology of primary uterine inertia (PUI), which is the most common cause of canine dystocia, is still not elucidated. Prostaglandins (PGs) play a crucial role in parturition. We hypothesized that the expression of prostaglandin endoperoxidase synthase 2 (PTGS2), PGF2α synthase (PGFS), and corresponding receptor (PTGFR) is altered in PUI. We investigated PTGS2, PGFS, and PTGFR mRNA expression, and PTGS2 and PGFS protein expression in interplacental (IP) and uteroplacental sites (UP) in bitches with PUI, obstructive dystocia (OD), and prepartum (PC). PTGS2, PGFS, and PTGFR mRNA expression did not differ significantly between PUI and OD (IP/UP). PTGFR ratio in UP was higher in PC than in OD (p = 0.014). PTGS2 immunopositivity was noted in foetal trophoblasts, luminal and superficial glandular epithelial cells, smooth muscle cells of both myometrial layers, and weakly and sporadically in deep uterine glands. PGFS was localized in luminal epithelial cells and in the epithelium of superficial uterine glands. PTGS2 and PGFS staining was similar between PUI and OD, while PGFS protein expression differed between OD and PC (p = 0.0215). For PTGS2, the longitudinal myometrial layer of IP stained significantly stronger than the circular layer, independent of groups. These results do not support a role for PTGS2, PGFS, and PTGFR in PUI. Reduced PGFS expression in IP during parturition compared with PC and the overall lack of placental PGFS expression confirm that PGFS is not the main source of prepartal PGF2alpha increase. The difference in PTGS2 expression between IP myometrial layers warrants further investigation into its physiological relevance.

A First Phenotypic and Functional Characterization of Placental Extracellular Vesicles from Women with Multiple Sclerosis.

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14+ monocytes and co-cultures of CD4+ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.

The distribution of some homeobox proteins in the bovine placenta during gestation

Homeobox proteins are transcription factors known to be involved in the molecular basics of body model formation and transformation. Some homeobox proteins are known to play critical roles in the control of the extraembryonic development of the placenta during gestation and in the regulation of uterine and placental physiology in adults. The gravid uteruses and placentas of 27 Holsteins cows, obtained from private slaughterhouses, were used in this study. The tissues were assigned to three groups as belonging to the first, second and third timesters of gestation, based on the fetal ages determined. Subsequently, the tissues were subjected to immunohistochemical staining using antibodies specific to the proteins investigated in the study. The evaluation of the immunohistochemical findings obtained in this study, demonstrated the presence of trimester-dependent varying intensities of immunoreactions in the uterus and placenta. Immunoreactivity was observed particularly in the luminal and glandular epithelial cells of the uterus, as well as in stromal and some endothelial cells. Furthermore, immunoreactivity for the proteins HOXA10, HOXB6, HOXC6 and Dlx-5 was determined in the smooth muscle cells. Moreover, immunoreactivity was also detected in the maternal epithelium and fetal trophoblasts found in the structure of the placenta. The results suggest that the homeobox proteins investigated may have critical roles in the regulation of endometrial functions in cows, and the proliferation and differentiation of endometrial and placental cells. It is concluded that these proteins may have physiological roles in the formation and development of the placenta, as well as in the maintenance of pregnancy.

Decidual and placental NOD1 is associated with inflammation in normal and preeclamptic pregnancies

IntroductionInflammation is a normal physiological process that increases to harmful levels in preeclampsia. It affects the interaction between maternal immune cells and fetal trophoblasts at both sites of the maternal-fetal interface; decidua and placenta. The pattern recognition receptor nucleotide-binding oligomerization domain-containing protein (NOD)1 is expressed at both sites. This study aimed to characterize the cellular expression and functionality of NOD1 at the maternal-fetal interface of normal and preeclamptic pregnancies. MethodsWomen with normal or preeclamptic pregnancies delivered by caesarean section were included. Decidual (n = 90) and placental (n = 91) samples were analyzed for NOD1 expression by immunohistochemistry and an automated image-based quantification method. Decidual and placental explants were incubated with or without the NOD1-agonist iE-DAP and cytokine responses measured by ELISA. ResultsNOD1 was markedly expressed by maternal cells in the decidua and by fetal trophoblasts in both decidua and placenta, with trophoblasts showing the highest NOD1 expression. Preeclampsia with normal fetal growth was associated with a trophoblast-dependent increase in decidual NOD1 expression density. Compared to normal pregnancies, preeclampsia demonstrated stronger correlation between decidual and placental NOD1 expression levels. Increased production of interleukin (IL)-6 or IL-8 after in vitro explant stimulation confirmed NOD1 functionality. DiscussionThese findings suggest that NOD1 contributes to inflammation at the maternal-fetal interface in normal pregnancies and preeclampsia and indicate a role in direct maternal-fetal communication. The strong expression of NOD1 by all trophoblast types highlights the importance of combined assessment of decidua and placenta for overall understanding of pathophysiological processes at the maternal-fetal interface.

Visualizing the Prevalence and Interactions of Maternal Immune Cells and Fetal Trophoblasts Using Imaging Mass Cytometry: The Prominence of Myeloid Cells

Visualizing the Prevalence and Interactions of Maternal Immune Cells and Fetal Trophoblasts Using Imaging Mass Cytometry: The Prominence of Myeloid Cells

Canine Endotheliochorial Placenta: Morpho-Functional Aspects.

In the domestic dog, placentation arises from central implantation, passing through a transitional, yet important stage of choriovitelline placenta (yolk sac placenta), on the way to the formation of the definite, deciduate, zonary (girdle) allantochorionic endotheliochorial placenta.Sharing some similarities with other invasive types of placentation, e.g., by revealing decidualization, it is characterized by restricted (shallow) invasion of trophoblast not affecting maternal capillaries and maternal decidual cells. Thus, being structurally and functionally placed between noninvasive epitheliochorial placentation and the more invasive hemochorial type, it presents an interesting and important model for understanding the evolutionarily determined aspects of mammalian placentation. More profound insights into the biological mechanisms underlying the restricted invasion of the fetal trophoblast into maternal uterine structures and the role of decidual cells in that process could provide better understanding of some adverse conditions occurring in humans, like preeclampsia or placenta accreta. As an important endocrine organ actively responding to ovarian steroids and producing its own hormones, e.g., serving as the source of gestational relaxin or prepartum prostaglandins, the canine placenta has become an attractive research target, both in basic and clinical research. In particular, the placental feto-maternal communication between maternal stroma-derived decidual cells and fetal trophoblast cells (i.e., an interplay between placenta materna and placenta fetalis) during the maintenance and termination of canine pregnancy serves as an interesting model for induction of parturition in mammals and is an attractive subject for translational and comparative research. Here, an updated view on morpho-functional aspects associated with canine placentation is presented.

IMMUNOHISTOCHEMICAL AND MORPHOMETRIC CHARACTERISTICS OF CHORIONIC TROPHOBLAST AND DECIDUAL CELLS OF FETAL MEMBRANES IN CASE OF ANTE-INTRANATAL FETAL DEATH

The aim: To identify immunohistochemical and morphometric features of chorionic trophoblast cells and fetal membranes derived decidual cells, which were obtained from stillbirths associated with pre-eclampsia, iron deficiency anemia, and acute chorioamnionitis. Materials and methods: The study included 58 fetal membranes of fetuses, who died in the ante-intranatal period. The membranes were divided into 6 obstetric history-based groups: premature (n = 8) and full-term (n = 8) stillbirths complicated by preeclampsia; premature (n = 8) and full-term ( n = 8) stillbirths complicated by iron deficiency anemia, premature (n = 10) and full-term (n = 16) stillbirths complicated by chorioamnionitis. A control group consisted of 8 membranes obtained from physiological pregnancies followed by the birth of a live full-term baby. Samples (rupture site) were probed with cytokeratin to identify the fetal trophoblast layer of the chorion and with vimentin for further identification of the decidual cells. The thickness of the trophoblastic layer, expression levels of cytokeratin and vimentin were determined. Results: A decrease of the cytokeratin expression by the chorionic trophoblasts and a thinning of the chorionic trophoblast cell layer due to an increasing gestational age were shown in case of the mentioned pathological conditions. In comparison with the control group, the level of vimentin expression by decidual cells was increased in case of full-term pregnancy complicated by preeclampsia, decreased in pregnancies complicated by chorioamnionitis, and remained unchanged in case of accompanying anemia. Conclusions: The changes in the studied immunohistochemical parameters are more pronounced in case of chorioamnionitis, which indicates more severe morphological and functional changes.

Shuni Virus Replicates at the Maternal-Fetal Interface of the Ovine and Human Placenta.

Shuni virus (SHUV) is a neglected teratogenic and neurotropic orthobunyavirus that was discovered in the 1960s in Nigeria and was subsequently detected in South Africa, Zimbabwe, and Israel. The virus was isolated from field-collected biting midges and mosquitoes and shown to disseminate efficiently in laboratory-reared biting midges, suggesting that members of the families Culicidae and Ceratopogonidae may function as vectors. SHUV infections have been associated with severe neurological disease in horses, a variety of wildlife species, and domesticated ruminants. SHUV infection of ruminants is additionally associated with abortion, stillbirth, and congenital malformations. The detection of antibodies in human sera also suggests that the virus may have zoonotic potential. To understand how SHUV crosses the ruminant placenta, we here infected pregnant ewes and subsequently performed detailed clinical- and histopathological examination of placental tissue. We found that SHUV targets both maternal epithelial cells and fetal trophoblasts, that together form the maternal-fetal interface of the ovine placenta. Experiments with human placental explants, furthermore, revealed replication of SHUV in syncytiotrophoblasts, which are generally highly resistant to virus infections. Our findings provide novel insights into vertical transmission of SHUV in sheep and call for research on the potential risk of SHUV infection during human pregnancies.

Transient Receptor Potential Vanilloid 6 (TRPV6) Proteins Control the Extracellular Matrix Structure of the Placental Labyrinth.

Calcium-selective transient receptor potential Vanilloid 6 (TRPV6) channels are expressed in fetal labyrinth trophoblasts as part of the feto–maternal barrier, necessary for sufficient calcium supply, embryo growth, and bone development during pregnancy. Recently, we have shown a less- compact labyrinth morphology of Trpv6-deficient placentae, and reduced Ca2+ uptake of primary trophoblasts upon functional deletion of TRPV6. Trpv6-/- trophoblasts show a distinct calcium-dependent phenotype. Deep proteomic profiling of wt and Trpv6-/- primary trophoblasts using label-free quantitative mass spectrometry leads to the identification of 2778 proteins. Among those, a group of proteases, including high-temperature requirement A serine peptidase 1 (HTRA1) and different granzymes are more abundantly expressed in Trpv6-/- trophoblast lysates, whereas the extracellular matrix protein fibronectin and the fibronectin-domain-containing protein 3A (FND3A) were markedly reduced. Trpv6-/- placenta lysates contain a higher intrinsic proteolytic activity increasing fibronectin degradation. Our results show that the extracellular matrix formation of the placental labyrinth depends on TRPV6; its deletion in trophoblasts correlates with the increased expression of proteases controlling the extracellular matrix in the labyrinth during pregnancy.

CXCR4 signaling at the fetal-maternal interface may drive inflammation and syncytia formation during ovine pregnancy†.

Early pregnancy features complex signaling between fetal trophoblast cells and maternal endometrium directing major peri-implantation events including localized inflammation and remodeling to establish proper placental development. Proinflammatory mediators are important for conceptus attachment, but a more precise understanding of molecular pathways regulating this process is needed to understand how the endometrium becomes receptive to implantation. Both chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal tissues. We identified this pair as a critical driver of placental angiogenesis, but their additional importance to inflammation and trophoblast cell survival, proliferation, and invasion imply a role in syncytia formation at the fetal-maternal microenvironment. We hypothesized that CXCL12 encourages both endometrial inflammation and conceptus attachment during implantation. We employed separate ovine studies to (1) characterize endometrial inflammation during early gestation in the ewe, and (2) establish functional implications of CXCL12 at the fetal-maternal interface through targeted intrauterine infusion of the CXCR4 inhibitor AMD3100. Endometrial tissues were evaluated for inflammatory mediators, intracellular signaling events, endometrial modifications, and trophoblast syncytialization using western blotting and immunohistochemistry. Endometrial tissue from ewes receiving CXCR4 inhibitor demonstrated dysregulated inflammation and reduced AKT and NFKB, paired with elevated autophagic activity compared to control. Immunohistochemical observation revealed an impairment in endometrial surface remodeling and diminished trophoblast syncytialization following localized CXCR4 inhibition. These data suggest CXCL12-CXCR4 regulates endometrial inflammation and remodeling for embryonic implantation, and provide insight regarding mechanisms that, when dysregulated, lead to pregnancy pathologies such as intrauterine growth restriction and preeclampsia.

Cholesterol Crystals and NLRP3 Mediated Inflammation in the Uterine Wall Decidua in Normal and Preeclamptic Pregnancies.

Preeclampsia is a hypertensive and inflammatory pregnancy disorder associated with cholesterol accumulation and inflammation at the maternal-fetal interface. Preeclampsia can be complicated with fetal growth restriction (FGR) and shares risk factors and pathophysiological mechanisms with cardiovascular disease. Cholesterol crystal mediated NLRP3 inflammasome activation is central to cardiovascular disease and the pathway has been implicated in placental inflammation in preeclampsia. Direct maternal-fetal interaction occurs both in the uterine wall decidua and at the placental surface and these aligned sites constitute the maternal-fetal interface. This study aimed to investigate cholesterol crystal accumulation and NLRP3 inflammasome expression by maternal and fetal cells in the uterine wall decidua of normal and preeclamptic pregnancies. Pregnant women with normal (n = 43) and preeclamptic pregnancies with (n = 28) and without (n = 19) FGR were included at delivery. Cholesterol crystals were imaged in decidual tissue by both second harmonic generation microscopy and polarization filter reflected light microscopy. Quantitative expression analysis of NLRP3, IL-1β and cell markers was performed by immunohistochemistry and automated image processing. Functional NLRP3 activation was assessed in cultured decidual explants. Cholesterol crystals were identified in decidual tissue, both in the tissue stroma and near uterine vessels. The cholesterol crystals in decidua varied between pregnancies in distribution and cluster size. Decidual expression of the inflammasome components NLRP3 and IL-1β was located to fetal trophoblasts and maternal leukocytes and was strongest in areas of proximity between these cell types. Pathway functionality was confirmed by cholesterol crystal activation of IL-1β in cultured decidual explants. Preeclampsia without FGR was associated with increased trophoblast dependent NLRP3 and IL-1β expression, particularly in the decidual areas of trophoblast and leukocyte proximity. Our findings suggest that decidual accumulation of cholesterol crystals may activate the NLRP3 inflammasome and contribute to decidual inflammation and that this pathway is strengthened in areas with close maternal-fetal interaction in preeclampsia without FGR.