Conventional natural killer cells control vascular remodeling in the uterus during pregnancy by acidifying the extracellular matrix with a2V.

Vascular remodeling within the uterus immediately before and during early pregnancy increases blood flow in the fetus and prevents the development of gestational hypertension. Tissue-resident natural killer (trNK) cells secrete pro-angiogenic growth factors but are insufficient for uterine artery (UtA) remodeling in the absence of conventional natural killer (cNK) cells. Matrix metalloproteinase-9 (MMP9) is activated in acidic environments to promote UtA remodeling. We have previously shown that ATPase a2V plays a role in regulating the function of cNK cells during pregnancy. We studied the effect of a2V deletion on uterine cNK cell populations and pregnancy outcomes in VavCrea2Vfl/fl mice, where a2V is conditionally deleted in hematopoietic stem cells. Conventional NKcells were reduced but trNK cells were retained in implantation sites at gestational day 9.5, and UtA remodeling was inhibited despite no differences in concentrations of pro-angiogenic growth factors. The ratio of pro-MMP9 to total was significantly elevated in VavCrea2Vfl/fl mice, and MMP9 activity was significantly reduced. The pH of implantation sites was significantly elevated in VavCrea2Vfl/fl mice. We concluded that the role of cNK cells in the uterus is to acidify the extracellular matrix (ECM) using a2V, which activates MMP9 to degrade the ECM, release bound pro-angiogenic growth factors, and contribute to UtA remodeling. Our results are significant for the understanding of the development of gestational hypertension.

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.

The curious case of vacuolar ATPase: regulation of signaling pathways.

The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.

Surfactant protein A suppresses preterm delivery induced by live Escherichia coli in mice.

Preterm birth accounts for the majority of neonatal morbidity and mortality in the developed world. A significant proportion of cases of spontaneous preterm labor are attributable to infections within gestational tissues. Surfactant protein A (SP-A), a collectin produced in the fetal lung and other tissues, has been shown previously in mice to suppress preterm delivery due to intrauterine (IU) instillation of sterile proinflammatory substances. Here we report a powerful antilabor effect for SP-A after IU infection with live Escherichia coli. SP-A abolished preterm birth (rate reduced from 100% to 0%) when it was administered into the uterus simultaneously with bacterial infection, reducing it by 75% when administered intravenously at the same time as IU bacterial inoculation, and by 48% when administered intravenously 4 h after IU bacterial infection. This effect on preterm delivery was accompanied by a parallel benefit on fetal survival in utero. SP-A had no effect on bacterial growth but reversed several major consequences of infection, including increased production of inflammatory mediators and a shift in macrophage polarization to the M1 phenotype. These findings suggest that exogenous SP-A has potential use to counteract infection-induced labor by reversing its proinflammatory consequences.

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.

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.

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.

1,25-Dihydroxy-vitamin D3 regulates NK-cell cytotoxicity, cytokine secretion, and degranulation in women with recurrent pregnancy losses.

Vitamin D has a pivotal role in regulating immune responses by promoting Th2 immune responses and suppressing Th1 responses. Propensities to a Th1 immune response and increased NK-cell levels and cytotoxicity have been reported in women with recurrent pregnancy losses (RPL). In women with RPL, vitamin D deficiency is prevalent; however, the effect of vitamin D on NK cells is largely unknown. In this study, we demonstrated that CD69(+) activating receptor expression on NK cells was significantly decreased by incubation with 1,25(OH)2 D3 in a dose-dependent manner, while CD158a and CD158b inhibitory receptor expression was upregulated. The degranulation marker CD107a was significantly downregulated on NK cells following incubation with 1,25(OH)2 D3 . NK-cell conjugation with K562 target cells was not affected by 1,25(OH)2 D3 ; however, depolarization of perforin granules in conjugated NK cells was significantly increased. TLR4 expression on NK cells was significantly decreased and TNF-α and IFN-γ production was significantly reduced by 1,25(OH)2 D3 through interference with NF-κB. Our results suggest 1,25(OH)2 D3 has immune regulatory effects on NK cell cytotoxicity, cytokine secretion and degranulation process as well as TLR4 expression. Potential therapeutic application of 1,25(OH)2 D3 for dysregulated NK-cell immunity should be explored in the future.

Regulation of apoptosis and innate immune stimuli in inflammation-induced preterm labor.

An innate immune response is required for successful implantation and placentation. This is regulated, in part, by the a2 isoform of V-ATPase (a2V) and the concurrent infiltration of M1 (inflammatory) and M2 (anti-inflammatory) macrophages to the uterus and placenta. The objective of the present study was to identify the role of a2V during inflammation-induced preterm labor in mice and its relationship to the regulation of apoptosis and innate immune responses. Using a mouse model of infection-induced preterm delivery, gestational tissues were collected 8 h after intrauterine inoculation on day 14.5 of pregnancy with either saline or peptidoglycan (PGN; a TLR 2 agonist) and polyinosinic-polycytidylic acid [poly(I:C); a TLR3 agonist], modeling Gram-positive bacterial and viral infections, respectively. Expression of a2V decreased significantly in the placenta, uterus, and fetal membranes during PGN+poly(I:C)-induced preterm labor. Expression of inducible NO synthase was significantly upregulated in PGN+poly(I:C)-treated placenta and uterus. PGN+poly(I:C) treatment disturbed adherens junction proteins and increased apoptotic cell death via an extrinsic pathway of apoptosis among uterine decidual cells and spongiotrophoblasts. F4/80(+) macrophages were increased and polarization was skewed in PGN+poly(I:C)-treated uterus toward double-positive CD11c(+) (M1) and CD206(+) (M2) cells, which are critical for the clearance of dying cells and rapid resolution of inflammation. Expression of Nlrp3 and activation of caspase-1 were increased in PGN+poly(I:C)-treated uterus, which could induce pyroptosis. These results suggest that the double hit of PGN+poly(I:C) induces preterm labor via reduction of a2V expression and simultaneous activation of apoptosis and inflammatory processes.

Platelet-activating factor: a role in preterm delivery and an essential interaction with Toll-like receptor signaling in mice.

Platelet-activating factor (PAF), a potent phospholipid activator of inflammation that signals through its cognate receptor (platelet-activating factor receptor, PTAFR), has been shown to induce preterm delivery in mice. Toll-like receptors (TLRs) are transmembrane receptors that mediate innate immunity. We have shown previously that Escherichia coli-induced preterm delivery in mice requires TLR signaling via the adaptor protein myeloid differentiation primary response gene 88 (MyD88), but not an alternative adaptor, Toll/IL-1 receptor domain-containing adapter protein-inducing interferon-beta (TRIF). In the present work, we analyzed the role of endogenously produced PAF in labor using mice lacking (knockout [KO]) PAF acetylhydrolase (PAF-AH; the key degrading enzyme for PAF). PAF-AH KO mice are more susceptible to E. coli-induced preterm delivery and inflammation than controls. In peritoneal macrophages, the PTAFR agonist carbamyl PAF induces production of inflammatory markers previously demonstrated to be upregulated during bacterially induced labor, including: inducible nitric oxide synthase (Nos2), the chemokine Ccl5 (RANTES), tumor necrosis factor (Tnf), and level of their end-products (NO, CCL5, TNF) in a process dependent upon both IkappaB kinase and calcium/calmodulin-dependent protein kinase II. Interestingly, this induced expression was completely eliminated not only in macrophages deficient in PTAFR, but also in those lacking either TLR4, MyD88, or TRIF. The dependence of PAF effects upon TLR pathways appears to be related to production of PTAFR itself: PAF-induced expression of Ptafr mRNA was eliminated completely in TLR4 KO and partially in MyD88 and TRIF KO macrophages. We conclude that PAF signaling plays an important role in bacterially induced preterm delivery. Furthermore, in addition to its cognate receptor, PAF signaling in peritoneal macrophages requires TLR4, MyD88, and TRIF.

Vitamin D deficiency may be a risk factor for recurrent pregnancy losses by increasing cellular immunity and autoimmunity.

STUDY QUESTION: Do women with recurrent pregnancy losses (RPL) and low vitamin D have increased prevalence of auto- and cellular immune abnormalities when compared with women with RPL who have normal vitamin D, and does vitamin D have any effect on cellular immunity in vitro? SUMMARY ANSWER: A high proportion of women with RPL have vitamin D deficiency and the risk of auto- and cellular immune abnormalities is increased in women with RPL and vitamin D deficiency. WHAT IS KNOWN ALREADY: Vitamin D deficiency in pregnant women is associated with increased risk of obstetrical complications such as pre-eclampsia, bacterial vaginosis associated preterm delivery, gestational diabetes mellitus and small-for-gestational age births. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional study of 133 women with RPL who were enrolled in a 2-year period, together with laboratory experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with three or more consecutive spontaneous abortions prior to 20 weeks of gestation who were enrolled at the University clinic. Serum vitamin D level, cellular activity and autoimmune parameters in vivo and in vitro were measured. MAIN RESULTS AND THE ROLE OF CHANCE: Sixty-three out of 133 women (47.4%) had low vitamin D (<30 ng/ml). The prevalence of antiphospholipid antibody (APA) was significantly higher in low vitamin D group (VDlow) (39.7%) than in the normal vitamin D group (VDnl) (22.9%) (P< 0.05) and the adjusted odds ratio (OR) for APA in VDlow was 2.22 with the 95% confidence interval (CI) of 1.0-4.7. The prevalence of antinuclear antigen antibody (VDlow versus VDnl; 23.8% versus 10.0%, OR 2.81, 95% CI 1.1-7.4), anti-ssDNA (19.0% versus 5.7%, OR 3.76, 95% CI 1.1-12.4) and thyroperoxidase antibody (33.3% versus 15.7%, OR 2.68, 95% CI 1.2-6.1) was significantly higher in VDlow than those of VDnl (P < 0.05 each). Peripheral blood CD19(+) B and CD56(+) NK cell levels and NK cytotoxicity at effector to target cell (E:T) ratio of 25:1 were significantly higher in VDlow when compared with those of VDnl (P < 0.05 each). Reduction (%) of NK cytotoxicity (at E:T ratio of 50:1 and 25:1) by IgG (12.5 mg/dl) was significantly lower in VDlow than those of VDnl (P < 0.05, P < 0.01, respectively). There were no differences in Th1/Th2 ratios between VDlow and VDnl. When vitamin D3 was added in NK cytotoxicity assay in vitro, NK cytotoxicity at E:T ratio of 50:1 was significantly suppressed with 10 nMol/L (nM) (11.9 ± 3.3%) and 100 nM (10.9 ± 3.7%) of vitamin D3 when compared with controls (15.3 ± 4.7%) (P < 0.01 each). TNF-α/IL-10 expressing CD3(+)/4(+) cell ratios were significantly decreased with 100 nM of vitamin D3 (31.3 ± 9.4, P < 0.05) when compared with controls (40.4 ± 11.3) in vitro. Additionally, INF-γ/IL-10 expressing CD3(+)/4(+) cell ratio was significantly decreased with 100 nM of vitamin D3 (12.1 ± 4.0, P < 0.05) when compared with controls (14.8 ± 4.6). IFN-γ and TNF-α secretion from NK cells were significantly decreased (P < 0.01 each), and IL-10, IL-1ß, vascular endothelial growth factor and granulocyte colony stimulating factor levels were significantly increased (P < 0.01 each) with vitamin D3 100 nM when compared with those of controls. LIMITATIONS, REASONS FOR CAUTION: The prevalence of vitamin D deficiency in women with RPL in this study is open to a possible type I error since women with vitamin D supplementation were excluded from this study. WIDER IMPLICATIONS OF THE FINDINGS: Assessment of vitamin D level is recommended in women with RPL. Vitamin D supplementation should be explored further as a possible therapeutic option for RPL. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the intramural funding from Department of Microbiology and Immunology, Chicago Medical School at Rosalind Franklin University of Medicine and Science. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Tumor-associated a2 vacuolar ATPase acts as a key mediator of cancer-related inflammation by inducing pro-tumorigenic properties in monocytes.

Cancer-related inflammation profoundly affects tumor progression. Tumor-associated macrophages (TAMs) are known regulators of that inflammation, but the factors that initiate cancer-related inflammation are poorly understood. Tumor invasiveness and poor clinical outcome are linked to increased expression of cell surface-associated vacuolar adenosine triphosphatases. The a2 isoform vacuolar adenosine triphosphatase is found on the surface on many solid tumors, and we have identified a peptide cleaved from a2 isoform vacuolar adenosine triphosphatase called a2NTD. a2NTD has properties necessary to induce monocytes into a pro-oncogenic TAM phenotype. The peptide upregulated both pro- and anti-inflammatory mediators. These included IL-1ß and IL-10, which are important in promoting inflammation and immune escape by tumor cells. The secretion of inflammatory cytokine IL-1ß was dependent on ATP, K(+) efflux, and reactive oxygen species, all mediators that activate the inflammasome. These findings describe a mechanism by which tumor cells affect the maturation of TAMs via a nontraditional cytokine-like signal, the a2NTD peptide.

V-ATPase upregulation during early pregnancy: a possible link to establishment of an inflammatory response during preimplantation period of pregnancy.

Various mechanisms exist to prevent a potentially deleterious maternal immune response that results in compromising survival of semiallogeneic fetus. In pregnancy, there is a necessary early preimplantation inflammatory stage followed by a postimplantation anti-inflammatory stage. Thus, there is a biphasic 'immune response' observed during the course of pregnancy. We provide the evidence that capacitation of sperm induced the expression of a2 isoform of V-ATPase (ATP6V0A2 referred to as a2V), leukemia inhibitory factor (Lif), Il1b, and Tnf in the sperm. Capacitated sperm also released cleaved N-terminal domain of a2V-ATPase (a2NTD), which upregulates the gene expression of Lif, Il1b, Tnf, and monocyte chemotactic protein-1 (Ccl2 (Mcp1)) in the uterus. Unfertilized eggs had low a2V expression, but after fertilization, the expression of a2V increased in zygotes. This increased level of a2V expression was maintained in preimplantation embryos. Seminal plasma was necessary for upregulation of a2V expression in preimplantation embryos, as mating with seminal vesicle-deficient males failed to elicit an increase in a2V expression in preimplantation embryos. The infiltration of macrophages into the uterus was significantly increased after insemination of both sperm and seminal plasma during the preimplantation period of pregnancy. This dynamic infiltration into the uterus corresponded with the uterine a2V expression through the induction of Ccl2 expression. Furthermore, the polarization ratio of M1:M2 (pro-inflammatory/anti-inflammatory) macrophages in the uterus fluctuated from a ratio of 1.60 (day 1) to 1.45 (day 4) when female mice were inseminated with both sperm and seminal plasma. These data provide evidence that exposure to semen may initiate an inflammatory milieu by inducing a2V and cytokine/chemokine expression, which triggers the influx of macrophages into the preimplantation uterus during the onset of pregnancy and ultimately leads to successful pregnancy outcome.

IL-22 regulates endometrial regeneration by enhancing tight junctions and orchestrating extracellular matrix.

The uterine endometrium uniquely regenerates after menses, postpartum, or after breaks in the uterine layer integrity throughout women's lives. Direct cell-cell contacts ensured by tight and adherens junctions play an important role in endometrial integrity. Any changes in these junctions can alter the endometrial permeability of the uterus and have an impact on the regeneration of uterine layers. Interleukin 22 (IL-22) is a cytokine that is recognized for its role in epithelial regeneration. Moreover, it is crucial in controlling the inflammatory response in mucosal tissues. Here, we studied the role of IL-22 in endometrial recovery after inflammation-triggered abortion. Fecundity of mice was studied in consecutive matings of the same animals after lipopolysaccharide (LPS) (10 µg per mouse)-triggered abortion. The fecundity rate after the second mating was substantially different between IL-22 knockout (IL-22-/-) (9.1%) and wild-type (WT) (71.4%) mice (p < 0.05), while there was no difference between the groups in the initial mating, suggesting that IL-22 deficiency might be associated with secondary infertility. A considerable difference was observed between IL-22-/- and WT mice in the uterine clearance following LPS-triggered abortion. Gross examination of the uteri of IL-22-/- mice revealed non-viable fetuses retained inside the horns (delayed clearance). In contrast, all WT mice had completed abortion with total clearance after LPS exposure. We also discovered that IL-22 deficiency is associated with a decreased expression of tight junctions (claudin-2 and claudin-10) and cell surface pathogen protectors (mucin-1). Moreover, IL-22 has a role in the remodeling of the uterine tissue in the inflammatory environment by regulating epithelial-mesenchymal transition markers called E- and N-cadherin. Therefore, IL-22 contributes to the proper regeneration of endometrial layers after inflammation-triggered abortion. Thus, it might have a practical significance to be utilized as a treatment option postpartum (enhanced regeneration function) and in secondary infertility caused by inflammation (enhanced barrier/protector function).

Ephrin-B2-expressing natural killer cells induce angiogenesis.

Background: Therapeutic angiogenesis aims to induce new blood vessel growth in ischemic tissues; however, previous clinical trials have had limited success. Studies of uterine angiogenesis revealed a specialized subset of natural killer (NK) cells, called uterine NK (uNK) cells, which have unique proangiogenic abilities. Methods: We show that uNK cells in mice express ephrin-B2, a regulator of angiogenesis, to induce tubule formation in an ex vivo coculture tubule formation assay. We next induced the expression of ephrin-B2 by splenic NK (sNK) cells harvested from male mice. Results: We showed that induced NK (iNK) cells can also instruct endothelial cells to form tubules using ephrin-B2. Conclusions: We concluded that Ephrin-B2 is a marker of proangiogenic uNK cells and that a proangiogenic phenotype characterized by ephrin-B2 can be induced in sNK cells to induce therapeutic angiogenesis.

Post-hoc evaluation of peripheral blood natural killer cell cytotoxicity in predicting the risk of recurrent pregnancy losses and repeated implantation failures.

Peripheral blood NK cytotoxicity assay (NKC) is one of the commonly utilized diagnostic tools for recurrent pregnancy losses (RPL) and repeated implantation failures (RIF). In this retrospective cohort study, we aimed to assess the cutoff values of NKC for RPL and RIF. A total of 883 women were included in this study; 24 nonpregnant fertile women, 604 nonpregnant women with three or more RPL, 163 nonpregnant women with two or more of RIF, 48 normal pregnant women, and 44 pregnant women with a history of RPL. Peripheral blood NKC assay was performed by flow cytometry. The differences between groups were analyzed using Student's t-test, a logistic regression analysis, and the area under the receiver operating characteristic curve analysis. Both nonpregnant fertile and normal pregnant women had significantly lower NKC at an effector to target cell ratio (E:T) of 50:1 (13.5 ± 1.1% and 12.9 ± 1.0%, respectively) when compared to women with RPL and RIF, and pregnant women with a history of RPL (23.6 ± 0.3%, 23.9 ± 0.5%, and 23.7 ± 1.0%, P < 0.0001 respectively). In addition, the area under the receiver operating characteristics curve for RPL and RIF using pre-conception NKC was 0.863 (P < 0.0001) and 0.879 (P < 0.0001), respectively, and for RPL using post-conception NKC was 0.736 (P = 0.001). These findings suggest that NKC significantly distinguishes nonpregnant women with RPL and RIF from fertile controls and pregnant RPLwomen from normal pregnant controls.

Placental ATPase expression is a link between multiple causes of spontaneous abortion in mice.

The a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a pivotal role in successful pregnancy and provides a microenvironment to maintain the delicate immunological balance at the feto-maternal interaction. We studied the expression of a2V mRNA in embryos and placenta of abortion-prone (female CBA × male DBA) murine matings or LPS (lipopolysaccharide)-treated mice. The expression of a2V was significantly higher in the placentas of nonabortion-prone (female BALB/c × male BALB/c and female CBA × male BALB/c) matings compared with the abortion-prone (female CBA × male DBA) mating. The expression of a2V was significantly decreased in the placentas treated with LPS in both female CBA × male DBA and female BALB/c × male BALB/c mating combinations with increased Lif, Il1b, and Tnf expression in the placenta. Decreased expression of a2V in the placenta is directly correlated with high percentages of pregnancy loss in abortion-prone mating (female CBA × male DBA) as well as in LPS-treated animals. The normal expression of placental a2V on Day 16 in the nonabortion-prone matings correlated with higher Mcp1 (monocyte chemotactic protein 1) gene expression, markedly higher infiltration of M1 and M2 macrophages, and no significant polarization patterns (M1/M2 = 1.2-1.6). However, in the abortion-prone mating, decreased placental a2V expression correlated with significantly lower Mcp1 gene expression with less infiltration of M1 and M2 macrophages and with polarization patterns skewed to M1 phenotypes (M1/M2 = 3.9-4.2). These data indicate that the higher expression of placental a2V is associated with dynamic infiltration of M1 and M2 macrophages through the induction of Mcp1 expression. This strengthens our hypothesis that a2V regulates the delicate cytokine and chemokine networks that coordinate the recruitment of macrophages for successful placental development and growth at the feto-maternal interface.

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.

Decidualization score identifies an endometrial dysregulation in samples from women with recurrent pregnancy losses and unexplained infertility.

OBJECTIVE: To study decidualization-associated endometrial factors. DESIGN: Retrospective cohort study to compare endometrial gene expression patterns in women experiencing reproductive failure including recurrent pregnancy loss or unexplained infertility versus fertile controls. SETTING: University Reproductive Medicine Center. PATIENTS: Women experiencing recurrent reproductive failure including recurrent pregnancy loss or unexplained infertility (n = 42) and fertile controls (n = 18). INTERVENTIONS: Endometrial biopsy samples were analyzed with targeted ribonucleic acid sequencing via next-generation sequencing. MAIN OUTCOME MEASURES: The primary end point measurements were the expression of genes important for endometrial transformation during decidualization measured singly and in a combined/cumulative score approach. The secondary end point measurements were receiver operating curve analysis and comparisons between the specific biomarkers. RESULTS: The comparison revealed differential expression of factors associated with decidualization, tissue homeostasis, and immune regulation: FOXO1, GZMB, IL15, SCNN1A, SGK1, and SLC2A1. A combined evaluation of these 6 signature factors was designated as a decidualization score in which the maximal score was "6" and the minimal was "0". Among controls, 89% of the samples had a score ≥5 and 11% had a score of "4". A total of 76% of samples in the patient group had scores ≤4 and 19% had the lowest score of "0". A decidualization score <4 provided evidence of abnormality in the decidualization process with a sensitivity of 76% (95% CI 61%-88%) and specificity of 89% (95% CI 65%-99%). CONCLUSIONS: Decidualization scoring can determine whether the endometrial molecular profile is implantation-friendly. Further validation of this testing approach is necessary to determine a particular patient population in whom it could be used for selecting patients that require therapeutic actions to improve endometrial conditions prior to the in vitro fertilization procedure.

Interleukin-22 promotes development of malignant lesions in a mouse model of spontaneous breast cancer.

Interleukin (IL)-22 is recognized as a tumor-supporting cytokine and is implicated in the proliferation of multiple epithelial cancers. In breast cancer, the current knowledge of IL-22 function is based on cell line models and little is known about how IL-22 affects the tumor initiation, proliferation, invasion, and metastasis in the in vivo system. Here, we investigated the tumor stage-specific function of IL-22 in disease development by evaluating the stage-by-stage progression of breast cancer in an IL-22 knockout spontaneous breast cancer mouse model. We found that among all the stages, IL-22 is specifically upregulated in tumor microenvironment (TME) during the malignant transformation stage of breast tumor progression. The deletion of IL-22 gene leads to the arrest of malignant transition stage, and reduced invasion and tumor burden. Administration of recombinant IL-22 in the TME does not influence in vivo tumor initiation and proliferation but only promotes malignant transformation of cancer cells. Mechanistically, deletion of IL-22 gene causes downregulation of epithelial-to-mesenchymal transition (EMT)-associated transcription factors in breast tumors, suggesting EMT as the mechanism of regulation of malignancy by IL-22. Clinically, in human breast tumor tissues, increased number of IL-22+ cells in the TME is associated with an aggressive phenotype of breast cancer. For the first time, this study provides an insight into the tumor stage-specific function of IL-22 in breast tumorigenesis.