Clinical molecular genetics evaluation in women with reproductive failures.

Molecular diagnostics is a rapidly growing branch of the clinical laboratory and has accelerated the advance of personalized medicine in the fields of pharmacogenomics, pharmacogenetics, and nutrigenomics. The versatility of molecular biology allows it to be effective in several medical fields that include reproduction, immunogenetics, and virology. Implementation of molecular and sequencing technology in reproductive medicine can add another layer of understanding to better define the causes behind infertility and recurrent reproductive loss. In the following, we examine current molecular methods for probing factors behind reproductive pregnancy loss including reverse transcription polymerase chain reaction and next generation sequencing (NGS). We review several current and potential genetic (DNA) and transcriptional (RNA)-based parameters in women with infertility that can be significant in diagnosis and treatment. These molecular factors can be inferred either from genomic DNA or RNA locally within the endometrium. Furthermore, we consider infection-based abnormalities such as human herpesvirus-6 and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Finally, we present future directions as well as data demonstrating the potential role of human endogenous retroviruses in pregnancy loss. We hope these discussions will assist the clinician in delineating some of the intricate molecular factors that can contribute to infertility and recurrent reproductive failures.

In vivo anti-V-ATPase antibody treatment delays ovarian tumor growth by increasing antitumor immune responses.

Tumor acidity is the key metabolic feature promoting cancer progression and is modulated by pH regulators on a cancer cell's surface that pump out excess protons/lactic acid for cancer cell survival. Neutralizing tumor acidity improves the therapeutic efficacy of current treatments including immunotherapies. Vacuolar-ATPase (V-ATPase) proton pumps encompass unique plasma membrane-associated subunit isoforms, making this molecule an important target for anticancer therapy. Here, we examined the in vivo therapeutic efficacy of an antibody (a2v-mAB) targeting specific V-ATPase-'V0a2' surface isoform in controlling ovarian tumor growth. In vitro a2v-mAb treatment inhibited the proton pump activity in ovarian cancer (OVCA) cells. In vivo intraperitoneal a2v-mAb treatment drastically delayed ovarian tumor growth with no measurable in vivo toxicity in a transplant tumor model. To explore the possible mechanism causing delayed tumor growth, histochemical analysis of the a2v-mAb-treated tumor tissues displayed high immune cell infiltration (M1-macrophages, neutrophils, CD103+ cells, and NK cells) and an enhanced antitumor response (iNOS, IFN-y, IL-1α) compared to control. There was marked decrease in CA-125-positive cancer cells and an enhanced active caspase-3 expression in a2v-mAb-treated tumors. RNA-seq analysis of a2v-mAb tumor tissues further revealed upregulation of apoptosis-related and toll-like receptor pathway-related genes. Indirect coculture of a2v-mAb-treated OVCA cells with human PBMCs in an unbuffered medium led to an enhanced gene expression of antitumor molecules IFN-y, IL-17, and IL-12-A in PBMCs, further validating the in vivo antitumor responses. In conclusion, V-ATPase inhibition using a monoclonal antibody directed against the V0a2 isoform increases antitumor immune responses and could therefore constitute an effective treatment strategy in OVCA.

Iodide Transporters in the Endometrium: A Potential Diagnostic Marker for Women with Recurrent Pregnancy Failures.

OBJECTIVE: The element iodine is an essential nutrient utilized by the thyroid glands, and deficiency of this element has been linked to reproductive failures. Iodide transporters are also present in reproductive tissues and cells of embryonic origin such as the endometrium and trophoblasts, respectively. The aim of this study is to understand if levels of iodide transporters are linked to pregnancy outcomes. SUBJECTS AND METHODS: RNA derived from endometrial biopsies from controls or women with recurrent reproductive failures was analyzed utilizing RT-PCR and targeted RNASeq. RESULTS: When compared to controls, women with 2 or more reproductive failures had a significant increase (>5 fold) in mRNA levels of the iodine transporters NIS and PENDRIN, but not thyroglobulin when probed vis RT-PCR. Targeted RNASeq analysis confirmed these findings when another group of patients were analyzed. CONCLUSION: These findings suggest possible abnormal iodine metabolism and a deficiency of iodine in endometrial tissues from some of the women with reproductive failures. We hypothesize from these findings that inorganic iodide and/or iodine is required for optimal cellular function in reproductive tissues, and that iodide transporters may potentially be used as a marker for infertility or for probing potential localized iodine deficiency that may not present in a typical thyroid panel analysis.

Poor ovarian response is associated with serum vitamin D levels and pro-inflammatory immune responses in women undergoing in-vitro fertilization.

Poor ovarian response (POR1) limits the success of infertility treatment modality. In this study, we aim to investigate if POR is associated with serum 25(OH) vitamin D (VD2) levels and pro-inflammatory immune responses in infertile women with a history of in-vitro fertilization and embryo transfer failures. A retrospective cross-sectional study included 157 women with IVF failures. Study patients were divided into four groups based on serum 25(OH)VD level and ovarian responses during the most recent IVF cycle; low VD (LVD3) with POR, LVD with normal ovarian response (NOR4), normal VD (NVD5) with POR, and NVD with NOR. Serum 25(OH)VD level, cellular- and auto-immunity, and metabolic parameters, including homocysteine and plasminogen activator inhibitor-1 were investigated. Peripheral blood CD56+ NK cell levels (%) and NK cytotoxicity were significantly higher in POR-LVD when compared to the other groups (P < 0.05, respectively). CD19 + B and CD19+/5+ B-1 cell levels were significantly higher in women with POR-LVD as compared with those of NOR-LVD and POR-NVD (P < 0.05, respectively). TNF-α/IL-10 producing Th1/Th2 cell ratio of POR-LVD was significantly higher than those of POR-NVD and NOR-NVD (P < 0.05 respectively). Peripheral blood homocysteine level of POR-LVD was significantly higher than those of NOR-LVD and POR-NVD (P < 0.05 respectively). We conclude that assessment of cellular and autoimmune abnormalities and metabolic factors, such as homocysteine should be considered in women with POR and LVD. VD and folic acid supplementation may be explored further as a possible therapeutic option for POR with immune and metabolic etiologies.

Ovarian and endometrial immunity during the ovarian cycle.

Immune tolerance is crucial for the successful pregnancy, while immune effectors and their products are required to safeguard a fetus from the infectious pathogens. The key immune effectors, such as T, B, and natural killer (NK) cells, monocytes, macrophages, and dendritic cells take part in regulating the immune responses at the maternal-fetal interface. The immune effectors become involved in intraovarian reproductive processes as well, such as ovulation, production of corpus luteum (CL) and its degeneration and determine the quality and evolution of the oocyte during the folliculogenesis. In the cycling endometrium, NK cells are rapidly infiltrated into the endometrium after ovulation and participate in angiogenesis and spiral artery remodeling process. In this study, we reviewed the characteristics and action mechanisms of immune effectors and their products in the peripheral blood, ovary, and endometrium during the ovarian cycle, since a comprehensive understanding of immune responses during the ovarian cycle and the time of implantation can help us to predict the pregnancy outcome and take effective measures for the prevention of potential obstetrical complications.

Mast cell-induced immunopathology in recurrent pregnancy losses.

PROBLEM: Mast cells (MC) have been known to play an important role in inflammation and angiogenesis by secreting numerous mediators, such as proteases, gelatinases, and proteoglycans. Three different MC subtypes were found in the endometrial layers of the uterus. In this study, we aim to investigate the role of endometrial MCs in recurrent pregnancy losses (RPL). METHOD OF STUDY: Endometrial biopsy was performed 5-7 days post-ovulation (implantation window) in women with a history of two or more RPL (n = 46) and normal fertile women (n = 10). Quantitative RT-PCR was performed to detect the expression of various mast cell mediators. Endometrial samples were evaluated using immunohistochemistry for c-kit receptor (CD117) and tryptase (MC activation marker). RESULTS: Mast cells were present throughout the entire layers of the endometrium; their count was elevated in RPL patients as compared to controls. The gene expression of c-Kit receptor was not different between the study groups. There are significant increases in the mRNA expression of various mediators, that is, stem cell factor (P = 0.029), tryptase (P = 0.024), heparan sulfate (P = 0.0005), and MMP-2 (P < 0.0001) in women with RPL as compared to normal controls. Chymase gene expression was not detected in most of the endometrial samples. CONCLUSION: This study has shown that MCs are overactive in RPL patients by creating a pro-inflammatory milieu, suggesting a novel role in the immunopathology of RPL. Future studies are needed to better understand the role of MC in implantation and placental angiogenesis.

Dysregulated uterine natural killer cells and vascular remodeling in women with recurrent pregnancy losses.

PROBLEM: Angiogenesis and vascular remodeling in secretory endometrium represent one of the crucial steps in pregnancy establishment, for which uterine NK (uNK) cells have an important role. Impairment of these steps may proceed to implantation and instigate initial pathology of recurrent pregnancy losses (RPL). In this study, we aim to investigate vascular development and density of uNK cells in secretory endometrium of women with RPL. METHODS OF STUDY: Mid-secretory phase endometrial tissues from women with RPL (n = 15) and fertile controls (n = 7) were investigated. CD56+ and CD16+ uNK cells, CD31+ vascular endothelial cells and smooth muscle myosin (SMM)+ . Vascular smooth muscle cells (VSMC) expressing SMM were investigated using immunohistochemistry and western blot. High-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was used as well. RESULTS: CD56+ uNK number was significantly higher in women with RPL compared to controls (P < 0.0001). uNK cell density by immunohistochemistry was positively correlated with CD56 mRNA expression by qRT-PCR (r2  = 0.43, P = 0.0137). The number of blood vessels represented by the expression of either CD31 or SMM was higher in women with RPL as compared to controls (P < 0.05 and P < 0.0001, respectively), and correlated with the number of uNK cell (r2  = 0.18, P < 0.04, and r2  = 0.65, P < 0.0001, respectively). The wall thickness of spiral arteries was significantly higher in women with RPL as compared with that of controls (P = 0.0027). CONCLUSION: Increased uNK cells in mid-secretory endometrium are associated with increased vascularization and defective vascular transformation of spiral arteries in women with RPL.

Inflammation induced preterm labor and birth.

Preterm birth which occurs before 37 weeks gestation is one of the most common obstetrical complication in humans. After many studies, it appears that "not one answer fits all" regarding the risk factors, causes and the treatments for this syndrome. However, it is becoming more evident that one of the major risk factors is inflammation and/or infection in the fetoplacental unit. In animal models (usually consisting of mice injected with lipopolysaccharide at 14 days of gestation), IL-22 and IL-6 have been identified as factors related to preterm birth. There are some clinical tests available to determine the risk for preterm labor and delivery, which can be identified before, during early, or at mid-gestation. However, treatment of preterm birth with antibiotics so far has not been "curable" and studies using anti-inflammatory treatments are not readily available. More studies regarding causes and treatments for preterm labor and delivery in humans are necessary to prevent neonatal deaths and/or developmental abnormalities associated with this common syndrome.

Interleukin 22 prevents lipopolysaccharide- induced preterm labor in mice.

Preterm birth is widespread and causes 35% of all neonatal deaths. Infants who survive face potential long-term complications. A major contributing factor of preterm birth is infection. We investigated the role of interleukin 22 (IL22) as a potential clinically relevant cytokine during gestational infection. IL22 is an effector molecule secreted by immune cells. While the expression of IL22 was reported in normal nonpregnant endometrium and early pregnancy decidua, little is known about uterine IL22 expression during mid or late gestational stages of pregnancy. Since IL22 has been shown to be an essential mediator in epithelial regeneration and wound repair, we investigated the potential role of IL22 during defense against an inflammatory response at the maternal-fetal interface. We used a well-established model to study infection and infection-associated inflammation during preterm birth in the mouse. We have shown that IL22 is upregulated to respond to an intrauterine lipopolysaccharide administration and plays an important role in controlling the risk of inflammation-induced preterm birth. This paper proposes IL22 as a treatment method to combat infection and prevent preterm birth in susceptible patients.

Mammary epithelium-specific inactivation of V-ATPase reduces stiffness of extracellular matrix and enhances metastasis of breast cancer.

Extracellular matrix (ECM) critically impacts tumor progression and is influenced by both cancer and host tissue cells. While our understanding of cancer cell ECM remodeling is widespread, the importance of host tissue ECM, which provides initial congenial environment for primary tumor formation, is partly understood. Here, we report a novel role of epithelial cell-associated vacuolar ATPase 'a2' isoform (a2V) in regulating breast tissue ECM stiffness to control metastasis. Using a mammary gland-specific a2V-knockout model, we show that in the absence of a2V, breast tumors exhibit atypically soft tumor phenotype, less tumor rigidity, and necrotic tumor microenvironment. These tumors contain a decreased number of cancer cells at primary tumor site, but showed extensive metastases compared to control. Nanomechanical evaluation of normal breast tissues revealed a decrease in stiffness and collagen content in ECM of a2V-deleted breast tissues. Mechanistically, inhibition of a2V expression caused dispersed Golgi morphology with relocation of glycosyltransferase enzymes to early endosomes in mammary epithelial cells. This resulted in defective glycosylation of ECM proteins and production of compromised ECM that further influenced tumor metastasis. Clinically, in patients with cancer, low a2V expression levels in normal breast tissue correlated with lymph node metastasis. Thus, using a new knockout mouse model, we have identified a2V expression in epithelial cells as a key requirement for proper ECM formation in breast tissue and its expression levels can significantly modulate breast tumor dissemination. Evaluation of a2V expression in normal breast tissues can help in identifying patients with high risk of developing metastases.

Identification of Novel Bisbenzimidazole Derivatives as Anticancer Vacuolar (H⁺)-ATPase Inhibitors.

The vacuolar (H⁺)-ATPases (V-ATPases) are a family of ATP-driven proton pumps and they have been associated with cancer invasion, metastasis, and drug resistance. Despite the clear involvement of V-ATPases in cancer, the therapeutic use of V-ATPase-targeting small molecules has not reached human clinical trials to date. Thus, V-ATPases are emerging as important targets for the identification of potential novel therapeutic agents. We identified a bisbenzimidazole derivative (V) as an initial hit from a similarity search using four known V-ATPase inhibitors (I-IV). Based on the initial hit (V), we designed and synthesized a focused set of novel bisbenzimidazole analogs (2a-e). All newly prepared compounds have been screened for selected human breast cancer (MDA-MB-468, MDA-MB-231, and MCF7) and ovarian cancer (A2780, Cis-A2780, and PA-1) cell lines, along with the normal breast epithelial cell line, MCF10A. The bisbenzimidazole derivative (2e) is active against all cell lines tested. Remarkably, it demonstrated high cytotoxicity against the triple-negative breast cancer (TNBC) cell line, MDA-MB-468 (IC50 = 0.04 ± 0.02 µM). Additionally, it has been shown to inhibit the V-ATPase pump that is mainly responsible for acidification. To the best of our knowledge the bisbenzimidazole pharmacophore has been identified as the first V-ATPase inhibitor in its class. These results strongly suggest that the compound 2e could be further developed as a potential anticancer V-ATPase inhibitor for breast cancer treatment.

Predicting NK cell subsets using gene expression levels in peripheral blood and endometrial biopsy specimens.

PROBLEM: In molecular analysis of tissue biopsy specimens, one crucial aspect is characterization of immune cell populations. This is especially important for evaluation of uterine receptivity by assessing levels of lymphocyte populations including CD56bright CD16- uterine NK cells and CD56dim CD16+ conventional NK cells. Our objective was to investigate whether measuring total RNA transcripts from a tissue specimen would accurately reflect immune cell levels and be a new technique to assess immune cell subsets. METHOD OF STUDY: Peripheral blood mononuclear cells (PBMCs) and endometrial tissues were used. Flow cytometry was utilized for the analysis of lymphocyte subsets in PBMCs, and RT-qPCR was applied to quantify RNA transcripts indicative of lymphocyte and granulocyte populations. RESULTS: In PBMC specimens, there were significant correlations between gene expression levels and cell subsets. NK cells correlated with CD16A, NKp46, and CD56 transcripts, B cells correlated with EBF1, and CD8+ T cells correlated with CD8ß. Finally, endometrial tissues displayed high CD56 expression and very low CD3ε, CD16A, and NKp30, reflecting the characteristic endometrial NK cell subsets. CONCLUSION: Strong correlations between RT-qPCR data and levels of lymphocyte subsets indicate that gene expression analysis will be a useful technique for characterizing levels of CD56+ cells in endometrial tissues.

The immune response in pregnancy and in cancer is active and supportive of placental and tumor cell growth not their destruction.

While many investigators have described the biochemical and physiological similarities between tumor cells and trophoblast cells, in this discourse we will compare primarily their leucocytes, which constitute a large portion of the tumor and its microenvironment as well as the placenta and its microenvironment. There is a remarkable similarity between the cells that support placental growth and development and tumor growth and development. In many cases over half of the cells present in the tumor and the placenta are non-tumor or nontrophoblast cells, immune cells. Most of these immune cells are prevented from attacking the fetal derived placental cells and the self-derived tumor cells. Nevertheless, these leucocytes, in our opinion, are very active and support tumor and placental cell growth through the production of growth factors and angiogenic factors. These cells do this by activating the portion of the immune response which initiates and helps control tissue repair.

Zika virus infection and biological treatment for reproductive medicine.

The recent Zika virus (ZIKV) epidemic is particularly challenging in the field of reproductive medicine as various biological tissues and byproducts, such as intravenous immunoglobulin G or cells are utilized during reproductive cycles, and an infected mother has an increased risk of having babies with fetal microcephaly and other congenital brain anomalies. In this review, current guidelines for prevention of sexual transmission of ZIKV, ZIKV testing, and tissue and blood product usages are summarized for physicians caring for those planning pregnancy or going through infertility treatment.

The V-ATPase a2 isoform controls mammary gland development through Notch and TGF-ß signaling.

Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of 'a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker ß-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-ß (transforming growth factor-ß) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-ß pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-ß are crucial for cell survival.

Pregnancy is a model for tumors, not transplantation.

Nearly 65 years have passed since Peter Medawar posed the following question: "How does the pregnant mother contrive to nourish within itself, for many weeks or months, a fetus that is an antigenically foreign body." Now, understanding of reproductive immunology has demonstrated that the HLA antigens in the placenta are non-classical and do not induce rejection. In the placenta and in tumors, 50% or more of the cells are cells of the immune system and were once thought to be primed and ready for killing tumors or the "fetal transplant" but these cells are not potential killers but abet the growth of either the tumor or the placenta. We believe that these cells are there to create an environment, which enhances either placental or tumor growth. By examining the similarities of the placenta's and tumor's immune cells, novel mechanisms to cause tumors to be eliminated can be devised.

Gonadotropin-releasing hormone analogues lead to pro-inflammatory changes in T lymphocytes.

PROBLEM: We aimed to investigate the effect of gonadotropin-releasing hormone (GnRH) analogues on T-cell immunity. METHOD OF STUDY: TNF-α(+) -, INF-É£(+) -, IL-10(+) -, and IL-17(+) -expressing T cells in peripheral blood mononuclear cells (PBMCs) were treated with various concentrations (0.1, 1, 5, and 10 µm) of GnRH agonist (buserelin acetate) and antagonist (cetrorelix acetate) for 4 hours in vitro and they were analyzed with flow cytometry. RESULTS: TNF-α(+) /IL-10(+) T helper (TH) cell ratios were increased in PBMCs treated with 1, 5, and 10 µm GnRH agonist when compared to controls (P=.006, P=.014 and P=.030, respectively). IFN-É£(+) /IL-10(+) TH cell ratios were significantly increased with 0.1, 1, 5, and 10 µm GnRH agonist as compared with controls (P=.046, P=.004, P=.013, and P=.011, respectively). TNF-α(+) TH cell levels, and IFN-γ(+) /IL-10(+) TH cell ratios were significantly different (P<.001 and P<.004, respectively) between GnRH agonist- and antagonist-treated cells. CONCLUSION: GnRH analogues induce pro-inflammatory TH1 shift in T-cell immunity, in vitro. GnRH treatment during assisted reproductive technology cycle might explain a possible cause of inflammatory flare in women with inflammatory conditions.

Selective inhibition of tumor cell associated Vacuolar-ATPase 'a2' isoform overcomes cisplatin resistance in ovarian cancer cells.

Development of resistance to platinum compounds significantly hinders successful ovarian cancer (OVCA) treatment. In tumor cells, dysregulated pH gradient across cell membranes is a key physiological mechanism of metastasis/chemo-resistance. These pH alterations are mediated by aberrant activation of key multi-subunit proton pumps, Vacuolar-ATPases (V-ATPases). In tumor cells, its 'a2' isoform (V-ATPase-V0a2) is a component of functional plasma-membrane complex and promotes tumor invasion through tumor-acidification and immuno-modulation. Its involvement in chemo-resistance has not been studied. Here, we show that V-ATPase-V0a2 is over-expressed in acquired-cisplatin resistant OVCA cells (cis-A2780/cis-TOV112D). Of all the 'a' subunit isoforms, V-ATPase-V0a2 exhibited an elevated expression on plasma membrane of cisplatin-resistant cells compared to sensitive counterparts. Immuno-histochemistry revealed V-ATPase-V0a2 expression in both low grade (highly drug-resistant) and high grade (highly recurrent) human OVCA tissues indicating its role in a centralized mechanism of tumor resistance. In cisplatin resistant cells, shRNA mediated inhibition of V-ATPase-V0a2 enhanced sensitivity towards both cisplatin and carboplatin. This improved cytotoxicity was mediated by enhanced cisplatin-DNA-adduct formation and suppressed DNA-repair pathway, leading to enhanced apoptosis. Suppression of V0a2 activity strongly reduced cytosolic pH in resistant tumor cells, which is known to enhance platinum-associated DNA-damage. As an indicator of reduced metastasis and chemo-resistance, in contrast to plasma membrane localization, a diffused cytoplasmic localization of acidic vacuoles was observed in V0a2-knockdown resistant cells. Interestingly, pre-treatment with monoclonal V0a2-inhibitory antibody enhanced cisplatin cytotoxicity in resistant cells. Taken together, our findings suggest that the isoform specific inhibition of V-ATPase-V0a2 could serve as a therapeutic strategy for chemo-resistant ovarian carcinoma and improve efficacy of platinum drugs.

Role of Notch signaling during lipopolysaccharide-induced preterm labor.

Notch signaling pathways exert effects throughout pregnancy and are activated in response to TLR ligands. To investigate the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligand Delta-like protein-1, transcriptional repressor hairy and enhancer of split-1, and Notch deregulator Numb were assessed. Preterm labor was initiated on gestation d 14.5 by 1 of 2 methods: 1) inflammation-induced preterm labor: intrauterine injection of LPS (a TLR4 agonist) and 2) hormonally induced preterm labor: subcutaneous injection of mifepristone. Delta-like protein-1, Notch1, and hairy and enhancer of split-1 were elevated significantly, and Numb was decreased in the uterus and placenta of inflammation-induced preterm labor mice but remained unchanged in hormonally induced preterm labor compared with their respective controls. F4/80(+) macrophage polarization was skewed in the uterus of inflammation-induced preterm labor toward M1-positive (CD11c(+)) and double-positive [CD11c(+) (M1) and CD206(+) (M2)] cells. This process is dependent on activation of Notch signaling, as shown by suppression of M1 and M2 macrophage-associated cytokines in decidual macrophages in response to γ-secretase inhibitor (an inhibitor of Notch receptor processing) treatment ex vivo. γ-Secretase inhibitor treatment also diminished the LPS-induced secretion of proinflammatory cytokines and chemokines in decidual and placental cells cultured ex vivo. Furthermore, treatment with recombinant Delta-like protein-1 ligand enhanced the LPS-induced proinflammatory response. Notch ligands (Jagged 1 and 2 and Delta-like protein-4) and vascular endothelial growth factor and its receptor involved in angiogenesis were reduced significantly in the uterus and placenta during inflammation-induced preterm labor. These results suggest that up-regulation of Notch-related inflammation and down-regulation of angiogenesis factors may be associated with inflammation-induced preterm labor but not with hormonally induced preterm labor.

Notch Signaling in Inflammation-Induced Preterm Labor.

Notch signaling plays an important role in regulation of innate immune responses and trophoblast function during pregnancy. To identify the role of Notch signaling in preterm labor, Notch receptors (Notch1-4), its ligands (DLL (Delta-like protein)-1/3/4), Jagged 1/2) and Notch-induced transcription factor Hes1 were assessed during preterm labor. Preterm labor was initiated on gestation day 14.5 by intrauterine (IU) injection of peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C). Notch1, Notch2, Notch4, DLL-1 and nuclear localization of Hes1 were significantly elevated in uterus and placenta during PGN+poly(I:C)-induced preterm labor. Ex vivo, Gamma secretase inhibitor (GSI) (inhibitor of Notch receptor processing) significantly diminished the PGN+poly(I:C)-induced secretion of M1- and M2-associated cytokines in decidual macrophages, and of proinflammatory cytokines (IFN-γ, TNF-α and IL-6) and chemokines (MIP-1ß) in decidual and placental cells. Conversely, angiogenesis factors including Notch ligands Jagged 1/2 and DLL-4 and VEGF were significantly reduced in uterus and placenta during PGN+poly(I:C)-induced preterm labor. In vivo GSI treatment prevents PGN+poly(I:C)-induced preterm delivery by 55.5% and increased the number of live fetuses in-utero significantly compared to respective controls 48 hrs after injections. In summary, Notch signaling is activated during PGN+poly(I:C)-induced preterm labor, resulting in upregulation of pro-inflammatory responses, and its inhibition improves in-utero survival of live fetuses.