Mouse Decidua Research Articles

Aberrant NK cell profile in gestational diabetes mellitus with fetal growth restriction.

Gestational diabetes mellitus (GDM) is a gestational disorder characterized by hyperglycemia, that can lead to dysfunction of diverse cells in the body, especially the immune cells. It has been reported that immune cells, specifically natural killer (NK) cells, play a crucial role in normal pregnancy. However, it remains unknown how hyperglycemia affects NK cell dysfunction thus participates in the development of GDM. In this experiment, GDM mice were induced by an intraperitoneal injection of streptozotocin (STZ) after pregnancy and it has been found that the intrauterine growth restriction occurred in mice with STZ-induced GDM, accompanied by the changed proportion and function of NK cells. The percentage of cytotoxic CD27-CD11b+ NK cells was significantly increased, while the proportion of nourished CD27-CD11b- NK cells was significantly reduced in the decidua of GDM mice. Likewise, the same trend appeared in the peripheral blood NK cell subsets of GDM patients. What's more, after intrauterine reinfusion of NK cells to GDM mice, the fetal growth restriction was alleviated and the proportion of NK cells was restored. Our findings provide a theoretical and experimental basis for further exploring the pathogenesis of GDM.

MiR-142-3p Regulates ILC1s by Targeting HMGB1 via the NF-κB Pathway in a Mouse Model of Early Pregnancy Loss.

Innate lymphoid cells (ILCs) are a class of newly discovered immunocytes. Group 1 ILCs (ILC1s) are identified in the decidua of humans and mice. High mobility group box 1 (HMGB1) is predicted to be one of the target genes of miR-142-3p, which is closely related to pregnancy-related diseases. Furthermore, miR-142-3p and HMGB1 are involved in regulating the NF-κB signaling pathway. This study aimed to examine the regulatory effect of miR-142-3p on ILC1s and the underlying mechanism involving HMGB1 and the NF-κB signaling pathway. Mouse models of normal pregnancy and abortion were constructed, and the alterations of ILC1s, miR-142-3p, ILC1 transcription factor (T-bet), and pro-inflammatory cytokines of ILC1s (TNF-α, IFN-γ and IL-2) were detected in mice from different groups. The targeting regulation of HMGB1 by miR-142-3p in ILC1s, and the expression of HMGB1 in normal pregnant mice and abortive mice were investigated. In addition, the regulatory effects of miR-142-3p and HMGB1 on ILC1s were detected in vitro by CCK-8, Annexin-V/PI, ELISA, and RT-PCR, respectively. Furthermore, changes of the NF-κB signaling pathway in ILC1s were examined in the different groups. For the in vivo studies, miR-142-3p-Agomir was injected in the uterus of abortive mice to evaluate the abortion rate and alterations of ILC1s at the maternal-fetal interface, and further detect the expression of HMGB1, pro-inflammatory cytokines, and the NF-κB signaling pathway. The number of ILC1s was significantly increased, the level of HMGB1 was significantly upregulated, and that of miR-142-3p was considerably downregulated in the abortive mice as compared with the normal pregnant mice (all P<0.05). In addition, miR-142-3p was found to drastically inhibit the activation of the NF-κB signaling pathway (P<0.05). The number of ILC1s and the levels of pro-inflammatory cytokines were significantly downregulated and the activation of the NF-κB signaling pathway was inhibited in the miR-142-3p Agomir group (all P<0.05). miR-142-3p can regulate ILC1s by targeting HMGB1 via the NF-κB signaling pathway, and attenuate the inflammation at the maternal-fetal interface in abortive mice.

Blastocyst-Derived Lactic Acid May Regulate S100A6 Expression and Function in Mouse Decidualization via Stimulation of Uterine Epithelial Arachidonic Acid Secretion.

(1) Background: Inflammatory responses are implicated in embryo implantation, decidualization, pregnancy maintenance and labor. Both embryo implantation and decidualization are essential to successful pregnancy in rodents and primates. S100A6 is involved in inflammation, tumor development, apoptosis and calcium homeostasis. S100A6 is strongly expressed in mouse decidua, but the underlying mechanisms of how S100A6 regulates implantation and decidualization are poorly defined. (2) Methods: Mouse endometrial stromal and epithelial cells are isolated from day 4 pseudopregnant mouse uteri. Both immunofluorescence and Western blotting are used to analyze the expression and localization of proteins. The molecular mechanism is verified in vitro by Western blotting and the quantitative polymerase chain reaction. (3) Results: From days 4 to 8 of pregnancy, S100A6 is specifically expressed in mouse subluminal stromal cells. Blastocyst-derived lactic acid induces AA secretion by activating the luminal epithelial p-cPLA2. The epithelial AA induces stromal S100A6 expression through the COX2/PGI2/PPAR δ pathway. Progesterone regulates S100A6 expression through the progesterone receptor (PR). S100A6/RAGE signaling can regulate decidualization via EGFR/ERK1/2 in vitro. (4) Conclusions: S100A6, as an inflammatory mediator, is important for mouse implantation and decidualization.

The regulation of tolerogenic dendritic cells by a Chinese herb formula improves abortion prone in mice.

Abortion prone (AP) is a common clinical event. The underlying mechanism remains unclear. Traditional Chinese formulas are known to be efficient in the management of abortion. The purpose of this study is to observe the effects of Anzitiaochongtang (AZT), a traditional formulation of Chinese medicine, on improving AP in mice by regulating immune tolerance. An established abortion model (CBA/J×DBA/2) was employed. AZT was prepared and administered to mice in a manner consistent with clinical practice. Tolerogenic dendritic cells (tDC) and type 1 regulatory T cells (Tr1 cell) in mice were analyzed by immunological approaches to be used as representative immune tolerant parameters. An AP model was established with CBA/J×DBA/2 mice. The expression of IL-10 in tDC and Tr1 cell frequency in the mouse decidua tissues were lower in the AP group than that in the normal pregnancy (NP) group. Administration of AZT up regulated the expression of IL-10 in tDCs and Tr1 cell generation in the decidua tissues, and improved the pregnancy and tissue structure in AP mice. The main mechanism by which AZT improves pregnancy in AP mice is that AZT enhanced the expression of galectin-9 in the epithelial cells of decidua tissues. Galectin 9 activates TIM3 on DCs to promote the IL-10 expression. The DCs induced more Tr1 cells in the decidua tissues. Dysfunctional tDCs were detected in the AP decidua tissues. Administration of AZT improved pregnancy in AP mice by regulating tDC function and generation of Tr1 cells in the maternal-fetal interface.

Stromal cells-specific retinoic acid determines parturition timing at single-cell and spatial-temporal resolution

The underlying mechanisms governing parturition remain largely elusive due to limited knowledge of parturition preparation and initiation. Accumulated evidences indicate that maternal decidua plays a criticalrole in parturition initiation. To comprehensively decrypt the cell heterogeneity in decidua approaching parturition, we investigate the roles of various cell types in mouse decidua process and reveal previously unappreciated insights in parturition initiation utilizing single-cell RNA sequencing (scRNA-seq). We enumerate the cell types in decidua and identity five different stromal cells populations and one decidualized stromal cells. Furthermore, our study unravels that stromal cells prepare for parturition by regulating local retinol acid (RA) synthesis. RA supplement decreases expression of extracellular matrix-related genes invitro and accelerates the timing of parturition invivo. Collectively, the discovery of contribution of stromal cells in parturition expands current knowledge about parturition and opens up avenues for the intervention of preterm birth (PTB).

Ovarian Stimulation in Mice Resulted in Abnormal Placentation through Its Effects on Proliferation and Cytokine Production of Uterine NK Cells.

Ovarian stimulation is associated with an increased incidence of abnormal placentation. Uterine natural killer (uNK) cells are the major subpopulation of decidual immune cells, which are crucial for placentation. In a previous study, we found that ovarian stimulation impairs uNK cell density on gestation day (GD) 8.5 in mice. However, it was not clear how ovarian stimulation led to a reduction in the density of uNK cells. In this study, we constructed two mouse models, an in vitro mouse embryo transfer model and an estrogen-stimulated mouse model. We used HE and PAS glycogen staining, immunohistochemical techniques, q-PCR, Western blot, and flow cytometry to analyze the mouse decidua and placenta, and the results showed that SO resulted in a fetal weight reduction, abnormal placental morphology, decreased placental vascular density, and abnormal density and function of uNK cells. Our results suggest that ovarian stimulation resulted in aberrant estrogen signaling and may contribute to the disorder of uNK cells caused by ovarian stimulation. Together, these results provide new insights into the mechanisms of aberrant maternal endocrine environments and abnormal placentation.

Expression and Regulation of a Novel Decidual Cells-Derived Estrogen Target during Decidualization.

During decidualization in rodents, uterine stromal cells undergo extensive reprogramming to differentiate into distinct cell types, forming primary decidual zones (PDZs), secondary decidual zones (SDZs), and layers of undifferentiated stromal cells. The formation of secondary decidual zones is accompanied by extensive angiogenesis. During early pregnancy, besides ovarian estrogen, de novo synthesis of estrogen in the uterus is essential for the progress of decidualization. However, the molecular mechanisms are not fully understood. Studies have shown that Cystatin B (Cstb) is highly expressed in the decidual tissue of the uterus, but the regulation and mechanism of Cstb in the process of decidualization have not been reported. Our results showed that Cstb was highly expressed in mouse decidua and artificially induced deciduoma via in situ hybridization and immunofluorescence. Estrogen stimulates the expression of Cstb through the Estrogen receptor (ER)α. Moreover, in situ synthesis of estrogen in the uterus during decidualization regulates the expression of Cstb. Silencing the expression of Cstb affects the migration ability of stromal cells. Knockdown Cstb by siRNA significantly inhibits the expression of Dtprp, a marker for mouse decidualization. Our study identifies a novel estrogen target, Cstb, during decidualization and reveals that Cstb may play a pivotal role in angiogenesis during mouse decidualization via the Angptl7.

Extracellular Vesicles Secreted by Mouse Decidual Cells Carry Critical Information for the Establishment of Pregnancy.

The mouse decidua secretes many factors that act in a paracrine/autocrine manner to critically control uterine decidualization, neovascularization, and tissue remodeling that ensure proper establishment of pregnancy. The precise mechanisms that dictate intercellular communications among the uterine cells during early pregnancy remain unknown. We recently reported that conditional deletion of the gene encoding the hypoxia-inducible transcription factor 2 alpha (Hif2α) in mouse uterus led to infertility. Here, we report that HIF2α in mouse endometrial stromal cells (MESCs) acts via the cellular trafficking regulator RAB27b to control the secretion of extracellular vesicles (EVs) during decidualization. We also found that Hif2α-regulated pathways influence the biogenesis of EVs. Proteomic analysis of EVs secreted by decidualizing MESCs revealed that they harbor a wide variety of protein cargoes whose composition changed as the decidualization process progressed. The EVs enhanced the differentiation capacity of MESCs and the production of angiogenic factors by these cells. We also established that matrix metalloproteinase-2, a prominent EV cargo protein, modulates uterine remodeling during decidualization. Collectively, our results support the concept that EVs are central to the mechanisms by which the decidual cells communicate with each other and other cell types within the uterus to facilitate successful establishment of pregnancy.

Endometrial stromal PRMT5 plays a crucial role in decidualization by regulating NF-κB signaling in endometriosis

Decidualization is a prerequisite for successful embryo implantation, in which elongated fibroblast-like endometrial stromal cells differentiate into more rounded decidual cells. Accumulating evidence has stressed the important role of the defective eutopic endometrium in infertility in endometriosis patients. However, the role of arginine methylation in the process of physiological decidualization and pathological decidualization defects is not clear. Here, we observed that the expression level of PRMT5, the main type II PRMT, was decreased in the endometrium of endometriosis patients, predominantly in stromal cells. Compared with the undecidualized state, PRMT5 was increased in the stromal cells of normal secretory endometrium in humans and in the decidua of normal pregnant mice or mice with artificially induced decidualization. The inhibition of PRMT5 resulted in a significant decrease in uterine weight and decidualization-related regulator expression, including FOXO1, HOXA10 and WNT4, in mice and IGFBP1 and prolactin levels in human endometrial stromal cells. Transcriptome analysis showed that decreased PRMT5 activity led to NF-κB signaling activation by inducing p65 translocation to the nucleus, which was also observed in endometriosis patients. Finally, overexpression of PRMT5 rescued the defective expression of IGFBP1 and prolactin in primary endometrial stromal cells from endometriosis patients. Our results indicate that promotion of PRMT5 may provide novel therapeutic strategies for the treatment of decidualization defects in infertile women, such as those with endometriosis.

Regulation and Function of Laminin A5 during Mouse and Human Decidualization.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

S04.1: The role of Nodal in modulating the inflammatory state in the mouse decidua

S04.1: The role of Nodal in modulating the inflammatory state in the mouse decidua

Stimulation of α7 Nicotinic Acetylcholine Receptor by Nicotine Suppresses Decidual M1 Macrophage Polarization Against Inflammation in Lipopolysaccharide-Induced Preeclampsia-Like Mouse Model.

Changes in decidual macrophage polarization affect local inflammatory microenvironment and lead to adverse pregnancy outcomes. However, the regulatory mechanism of macrophage polarization in preeclampsia (PE) remains unclear. In this study, we found that α7nAChR expression was significantly down-regulated in decidual macrophages in PE patients compared to normal pregnant women, accompanied by a reduced proportion of M2 phenotype and an increased proportion of M1 phenotype; these results suggested that the reduced α7nAChR activity might contribute to changes in the polarization of decidual macrophages. Then, we further investigated the regulatory role of α7nAChR activation by nicotine on decidual macrophage polarization and placental remodeling in the PE-like mouse model. The PE mice were obtained by i.p. injection of 10 µg/kg lipopolysaccharide (LPS) gestational day (GD) 13, and 40 µg/kg LPS daily until GD16. Subcutaneous injection of 1.0 mg/kg nicotine was administrated from GD14 to GD18. Nicotine treatment increased the decreased M2 phenotype and inhibited the increased M1 phenotype in decidua of pregnant mice induced by LPS. The levels of pro-inflammatory cytokines in decidua were higher but the levels of anti-inflammatory cytokines were lower in PE mice compared to the controls, nicotine reversed these changes. The level of choline acetyltransferase (CHAT) was reduced in the LPS-treated group, it was increased following nicotine treatment. Damage of spiral artery remodeling and down-regulation of markers related to trophoblast invasion in placentas were found in PE mice; nicotine improved these pathological structures of placentas. α-bungarotoxin (α-BGT) which is specific antagonist for α7nAChR could abolish the effects of nicotine on decidual macrophage polarization, trophoblast arrangement and vascular structure in placental tissue in PE mice. These results suggest that α7nAChR plays an important regulatory role in maternal-fetal inflammation and placental remodeling in preeclampsia and may provide a theoretical basis for the discovery of new strategies for preeclampsia.

Bone-marrow-derived endothelial progenitor cells contribute to vasculogenesis of pregnant mouse uterus†.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n=5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

Comparative analysis of decidual and peripheral immune cells and immune-checkpoint molecules during pregnancy in wild-type and PACAP-deficient mice.

PACAP is a neuropeptide having a major relevance in the nervous system and in several peripheral organs including those of the reproductive system. PACAP-deficient mice have several morphological, biochemical, behavioral defects, and show reduced fertility. Female reproductive functions such as fertility, mating behavior, maternal behaviors, and implantation alterations have been widely investigated, but no comparative immune analyses are available in pregnant wild-type (WT) and PACAP knockout (KO) mice. Therefore, we performed a detailed immunophenotyping of decidual and peripheral immune cells and investigated the expression of two immune-checkpoint molecules by immune cells together with immunohistochemistry detecting Galectin-9 in placental tissues. We investigated the percentage of numerous immune cell populations in the periphery and in the decidua of pregnant mice. We demonstrated a significant increase in the frequency of decidual Gal-9+ Th cells obtained from PACAP KO mice compared to the decidua of WT mice. We could not determine statistical differences in TIM-3 and programmed cell death-1 expression by different immune cells in the decidua and in the periphery between WT and KO mice. In conclusion, we could not find any significant alteration either in the distribution or in the cytotoxicity of the investigated decidual immune cells which could elucidate any reproductive alterations in PACAP KO mice. The only remarkable finding is the recruitment of Gal-9+ Th cells to the decidua promoting local immune homeostasis in PACAP KO mice, which nevertheless cannot explain the reduced fertility observed in these mice.

Effect and mechanism of Yunkang oral liquid in regulating endocrine system and VEGF signaling pathway and reducing abortion rate in recurrent abortion mice

This experiment focuses on the effect of Yunkang oral liquid on abortion rate, endocrine system and VEGF signal pathway in Clark classical recurrent abortion model mice. RSA mice were randomly divded into model group, low, middle and high-dose groups and progesterone group. The normal pregnancy mice were included into normal group. Since the first day of pregnancy, the normal group and the RSA model group were given the same dose of distilled water, while low, middle and high-dose groups were given Yunkang oral liquid at the dose of 9, 18, 36 mL·kg¹·d⁻¹; progesterone group were given progesterone by 0.039 g·kg¹·d⁻¹. The mice were put to deathat the 15th day of pregnancy, and the embryo loss rate of each group was observed. Serum estradiol (E₂), progesterone (P), prolactin (PRL), luteinizing hormone (LH), follicle stimulating hormone (FSH) level were tested; the protein expressions of estrogen receptor(ER), progesterone receptor (PR), prolactin receptor (PRLR) in decidua and RAS, MAPK, VEGF, VEGFR-2 gene and protein expressions in deciduas were studied. The results showed that middle, high dose Yunkang and progesterone could significantly decrease the embryo loss rate of RSA mice. The levels of FSH, LH, PRL, P and E₂ in serum in Yunkang and progesterone groups were increased, and the serum levels of FSH, LH, and E₂ in Yunkang group were higher than those in progesterone group. Western blot analysis showed that Yunkang oral liquid and progesterone can significantly increase the expressions of PRLR, PR in the uterine decidua of RSA mice, and the expression of ER in Yunkang group was higher than that in progesterone group. Western blot and PCR showed that the Yunkang oral liquid and progesterone can significantly increase RAS, MAPK, VEGF, VEGFR-2 gene and protein expressions in the uterine decidua of RSA mice. The results showed that Yunkang oral liquid can effectively reduce the embryo loss rate of RSA model mice, increase the levels of FSH, LH, PRL, P and E₂ in serum, promote the expressions of PRLR, PR, ER protein in decidua and the RAS, MAPK, VEGF, VEGFR-2 gene and protein expressions in the decidua, improve the vascular remodeling of fetal interface, the endometrial receptivty, the development of decidua and the blastocyst implantation.

Neutrophil extracellular traps are critical for pregnancy loss in sphingosine kinase-deficient mice on 129Sv/C57BL/6 background.

Exaggerated maternal immune responses must be strictly controlled to ensure a successful pregnancy. Neutrophil extracellular traps (NETs) have recently been implicated as a potential mechanism for promoting inflammation in pregnancy-related disorders. In this study, we demonstrated that NETs play a key role in the pathogenesis of sphingosine kinase (Sphk)-mediated pregnancy loss. Perturbing the sphingolipid pathway by disrupting Sphk genes during pregnancy led to excessive NET formation exclusively at the fetomaternal interface and early fetal death. Neutrophils that formed NETs were characterized by histone hypercitrullination and peptidylarginine deiminase 4 (PAD4) overexpression. In addition, thrombus formation was enhanced in the decidua, but not in the plasma, of Sphk-deficient mice. Blocking NET formation with a PAD4 inhibitor protected Sphk-deficient mice from pregnancy loss. The PAD4 inhibition significantly reduced the expression of hypercitrullinated histone in neutrophils and ameliorated vascular injury in the decidua of Sphk-deficient mice. Moreover, NET formation was induced in human neutrophils stimulated with Sphk-deficient human decidual cells. Together, these findings indicate that targeting NETs might be a novel therapeutic strategy to treat idiopathic pregnancy loss in humans.-Mizugishi, K., Yamashita, K. Neutrophil extracellular traps are critical for pregnancy loss in sphingosine kinase-deficient mice on 129Sv/C57BL/6 background.

Distinct effects of short- and long-term type 1 diabetes to the placental extracellular matrix and fetal development in mice

PurposeWe have previously shown that the development of complications in the early pregnant decidua and myometrium in mice correlates with diabetes progression. In the current study, we investigated the influence of diabetes progression on the placental extracellular matrix (ECM) and on fetal development at the end of pregnancy. MethodsAlloxan-induced type 1 diabetic female mice were bred either 30–50 days after diabetes induction (D) or 90-110D. Fetal and placental weights were registered at the 19th day of pregnancy together with analysis of gene expression, deposition and turnover of the placental ECM. ResultsThe short-term diabetic group (30-50D) showed elevated embryonic losses and underweight fetuses (89%) with normal weight placentas. In contrast, the long-term group (90-110D) had increased malformations/fetal deaths and underweight fetuses (42%) and heavy placentas (50%). Normal-weight fetuses from the long-term group had placentas with either regular weight and fetal/placental weight ratio or increased weight and low fetal/placental weight ratio. Furthermore, the placentas of the short-term group showed alterations in the synthesis and deposition of collagen types I and V and in the activity of MMP2 whereas placentas of the 90-110D group presented alterations in collagen type III and V and MMP9. ConclusionsDiabetes progression promoted distinct outcomes in pregnancy. Modifications of both synthesis and turnover of ECM occurred even before changes of placental weight were detected. Adjustment of fetal/placental weight ratio or placental enlargement restored normal growth in part of the fetuses from the long-term group.

Natural cytotoxicity receptor 1 in mouse uNK cell maturation and function.

Early and midgestational decidua of mice genetically ablated for expression of the natural killer (NK) cell natural cytotoxicity receptor (NCR; Ncr1Gfp/Gfp mice) shows restricted angiogenesis and atypically small uterine (u)NK cells. We hypothesized that NCR1 inactivation disturbs maturation and angiokine production by uterine natural killer (uNK) cells. Using histological and morphometric approaches, we observed that Ncr1Gfp/Gfp but not control C57BL/6 (B6) implantation sites sustain immature, non-granulated uNK cells into midpregnancy. Mouse uNK cells can be subclassified by their reactivity with Dolichos biflorus agglutinin (DBA) lectin; DBA+ uNK cells with greater Ncr1 expression were investigated. DBA+ uNK cells from Ncr1Gfp/Gfp mice show delayed maturation as indicated by shorter diameters and fewer cytoplasmic granules. Granules in mature Ncr1Gfp/Gfp uNK cells are ultrastructurally abnormal and abundance of granule-associated proteins (perforin, granzyme) and of cytoplasmic proteins (vascular endothelial growth factor; placental growth factor) differs from controls. Leukocyte-leukocyte conjugate formation in gestation day 6.5 and 8.5 intact Ncr1Gfp/Gfp decidua was less frequent than in B6; however, this difference involved leukocytes other than DBA+ uNK cells. These studies strongly support roles for NCR1 and its ligands in normal pregnancy promotion.

Immune regulatory network in successful pregnancy and reproductive failures.

Maternal immune system must tolerate semiallogenic fetus to establish and maintain a successful pregnancy. Despite the existence of several strategies of trophoblast to avoid recognition by maternal leukocytes, maternal immune system may react against paternal alloantigenes. Leukocytes are important components in decidua. Not only T helper (Th)1/Th2 balance, but also regulatory T (Treg) cells play an important role in pregnancy. Although the frequency of Tregs is elevated during normal pregnancies, their frequency and function are reduced in reproductive defects such as recurrent miscarriage and preeclampsia. Tregs are not the sole population of suppressive cells in the decidua. It has recently been shown that regulatory B10 (Breg) cells participate in pregnancy through secretion of IL-10 cytokine. Myeloid derived suppressor cells (MDSCs) are immature developing precursors of innate myeloid cells that are increased in pregnant women, implying their possible function in pregnancy. Natural killer T (NKT) cells are also detected in mouse and human decidua. They can also affect the fetomaternal tolerance. In this review, we will discuss on the role of different immune regulatory cells including Treg, γd T cell, Breg, MDSC, and NKT cells in pregnancy outcome.

PIBF positive uterine NK cells in the mouse decidua

Though uterine NK cells (u NK cells) contain cytotoxic granules, and selectively over- express the genes of perforin and granzymes, during normal pregnancy, they are not cytotoxic.Progesterone is indispensable for the establishment and maintenance of pregnancy both in humans and in mice. Mouse uterine NK cells do not express the classical progesterone receptor, yet progesterone affects the recruitment and function of uterine NK cells, the latter partly via the Progesterone-Induced Blocking Factor (PIBF). We demonstrated PIBF positive granulated cells in the mouse decidua. The aim of this study was to characterize these cells by lectin immunohistochemistry and anti-perforin reactivity.PIBF+ granulated cells were absent from the deciduae of alymphoid mice, but appeared in the decidua of those that had been reconstituted with bone marrow from male BALB/c mice. PIBF+ granulated cells bound the DBA lectin, suggesting their NK cell nature, and also contained perforin, which co-localized with PIBF in the cytoplasmic granules. In anti-progesterone treated mice all of the PIBF+ cells were perforin positive at g. d. 12.5, in contrast to the 54% perforin positivity of PIBF+ cells in untreated mice. ConclusionThe PIBF+ granulated cells in the decidua belong to the NK population, and PIPB co-localizes with perforin in the cytoplasmic granules.