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.

BACKGROUND:A number of laboratory tests have been developed to determine properties of spermatozoa quality but few have been adopted into routine clinical use in place of the WHO semen analysis. We investigated whether Atp6v0a2 (a2 isoform of vacuolar ATPase) is associated with abnormal semen quality and changes in chemokine-cytokine profiles in infertile men.PATIENTS AND METHODS:Semen samples were collected from 35 healthy donors and 35 infertile men at the Andrology laboratory from August 2011 to June 2012. The levels of Atp6v0a2 mRNA and protein, and its localization in spermatozoa were determined. a2NTD (the N-terminal portion of Atp6v0a2) and secreted chemokine-cytokine profiles in seminal fluid were measured.RESULTS:Atp6v0a2 protein (P<0.05) and mRNA (P<0.05) in spermatozoa from infertile men were significantly lower than those from fertile men. Fluorescent microscopy revealed that Atp6v0a2 is mainly expressed in the acrosomal region. Infertile men's seminal fluid had significantly lower G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) levels when compared to the seminal fluid from fertile men. Seminal fluid a2NTD levels were significantly correlated with G-CSF (P<0.01), GM-CSF (P<0.01), MCP-1 (P<0.05), MIP-1α (P<0.01) and TGF-β1 (P<0.01) which are key molecules during the onset of pregnancy.CONCLUSION:These results suggested that a critical level of Atp6v0a2 is required for the fertile spermatozoa and its decreased level in spermatozoa could be used to predict male infertility. This study provides a possibility that Atp6v0a2 could be potentially used as a diagnostic marker for the evaluation of male infertility.

Summary The objective of the present study was to investigate the effect of Gram-negative bacteria infection on ovarian steroid receptors, i.e. progesterone receptor (PR) and estradiol receptor (ER) during preimplantation days of pregnancy. A well established mouse model of Gram-negative bacteria infection was used to test this objective. Mice were treated with normal saline or lipopolysaccharide (LPS) on day 0.5 of pregnancy and used to collect embryos and uterine horns on day 1.5 to day 4.42 preimplantation day of pregnancy. Total RNA was extracted and reverse-transcription polymerase chain reaction (PCR) was performed to check the expression of PR and ER genes. The mRNA expression of PR and ER was altered in embryos and uterus of LPS-treated animals during preimplantation days of pregnancy studied. These results suggest that PR and ER play an important role in Gram-negative bacteria infection and induced implantation failure in mouse.

The objective of the present study is to investigate the role of heat shock proteins (Hsps) in preimplantation embryonic development and uterine receptivity during lipopolysaccharide (LPS)-induced pregnancy loss. Mice were treated with PBS or LPS on Day 0.5 of pregnancy, and preimplantation embryos and uterus were collected on Days 1.5-4.42 of pregnancy. The individual preimplantation embryos were assessed for their morphologic appearance and DNA damage during the preimplantation period of pregnancy. The expression of Hsp90, Hsp70, Hsp60, and Hsp25 was determined in preimplantation embryos and uterus by RT-PCR. Comet studies showed that LPS treatment significantly increased the percentage of abnormal embryos and DNA damage in the embryos. The expression of Hsp90, Hsp70, and Hsp60 was significantly lower in preimplantation embryos recovered from LPS-treated mice when compared to their respective controls. The expression of Hsp90, Hsp70, Hsp60, and Hsp25 was altered in uterus of LPS-treated mice when compared to their respective controls. Immunohistochemistry studies showed that at the time of implantation (i.e., Day 4.42), levels of Hsp90 and Hsp60 were decreased in stromal cells of LPS-treated uterus when compared to their respective controls. Hsp25 was highly expressed in the endometrium and stromal cells of LPS-treated uterus. Our results clearly showed that lowering of embryonic expression of Hsps induces DNA damage, which leads to degeneration and degradation of preimplantation embryos, and altered uterine expression of Hsps may not prepare the uterus for implantation. This may ultimately lead to implantation failure in mouse.

Purpose The purpose of the present study was to investigate the impact of lipopolysaccharide (LPS) on follicle-stimulating hormone (FSH), luteinizing hormone (LH) and their receptors during preimplantation days of pregnancy. Method The PBS or lipopolysaccharide (LPS) was injected intraperitoneally in the pregnant females on day 0.5 of pregnancy and serum, embryos, ovaries and uterine horns were collected on days 1.5, 2.5, 3.5, 4.0, 4.125, 4.33 and 4.42 of pregnancy. Result(s) In the LPS-treated pregnant females, the secretion of FSH and LH is disturbed with respect to normal pregnancy. Furthermore, the expression of FSHR mRNA in embryos and ovaries, LHR mRNA in embryos and uterus get modulated in response to LPS during preimplantation days of pregnancy. Conclusion(s) The disturbance in the serum level of FSH and LH in response to LPS leads implantation failure in mouse which suggests that these gonadotropins plays an integral role in the process of the successful implantation. This study also suggests a possible nongonadal role of FSHR and LHR in LPS-induced implantation failure in the mouse.

OBJECTIVE Polymyxin B (PB) is a naturally occurring cationic cyclic decapeptide which is highly bactericidal to Gram-negative bacteria. The objective of this study was to investigate the effect of PB on the viability of developing embryos during pregnancy and to validate its protective effect on the embryotoxic effect of Gram-negative bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS Animals were injected intraperitoneally (i.p.) with PB (5-100 μg/animal), (Minimum effective dose) MD of LPS and MD of LPS+PB (5-100 μg/animal) on day 0.5 of pregnancy. The percentage of normal gestational sacs and histopathologic analysis were assessed. RESULTS PB treatment of pregnant females disturbs the pregnancy in a dose dependent manner and increases the substantial risk of congenital abnormalities in the growing fetuses of the mother. However, PB does not show any adverse effect on implantation of embryos. The embryotoxic effect of LPS can be prevented completely by 25 μg PB/animal; however other lower and higher doses of PB were not able to protect against the effect of LPS on pregnancy. CONCLUSIONS Our results demonstrate that PB has the ability to protect the LPS-induced pregnancy loss but may not be recommended as a safe drug for the treatment of a mother suffering from Gram-negative bacterial infection during pregnancy.

Our objective was to investigate the effect of gram-negative bacterial infection on the ovaries and serum level of P-4 and 17 beta-E-2 during the preimplantation days of pregnancy in the mouse. We found that lipopolysaccharide alters the serum level of P-4 and E-2 during the preimplantation days of pregnancy and elevates the E-2/P-4 ratio, which may keep the uterus nonreceptive during the preimplantation days of pregnancy and also not prepare the developing blastocysts for implantation in the mouse. A large infiltration of macrophages in the corpora lutea and appearance of graafian follicles from day 3.5 of pregnancy because of lipopolysaccharide treatment, which reflect a gram-negative bacterial infection, may be responsible for ovarian dysfunction and altered P-4 and E-2 level in serum. (Fertil Steril (R) 2011;95:1471-4. (C)2011 by American Society for Reproductive Medicine.)

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.

The a2 isoform of vacuolar ATPase (A2V-ATPase) has immunosuppressive effects and is in part responsible for the survival of the fetal allograft. Pregnancy brings vital changes in the maternal environment that comprises reversible modifications of immune cells in response to both systemic and local mediators and signals. The spleen can be a useful organ to determine the systemic affects of pregnancy on multiple cell populations, including those of the immune system. By taking the well studied, allogeneic abortion prone (maleCBAX femaleDBA) model and other syngeneic and allogeneic mating models, the effects of pregnancy on murine immune cells of the spleen were studied. We assessed the expression of A2V-ATPase in the spleen of different mating combinations on different days of pregnancy. Our data shows that the splenic A2V-ATPase gene expression increased from day 8 to 16 in maleBALB/cXfemaileBALB/c and maleCBA XfemaleBALB/c matings established as successful pregnancy controls. However, in abortion prone mating (maleCBAX femaleDBA) its expression was similar as the successful pregnancy controls on day 8 and then decreased significantly on later days of pregnancy. Additionally, administration of LPS at different days of pregnancy significantly decreased the expression of A2V-ATPase in spleen. We also assessed the surface expression of A2V-ATPase on splenic lymphocytes at different days of pregnancy by using flow cytometry. We show that the expression of A2V-ATPase in CD4+ and CD8+ cells was significantly lower in both abortive prone mating as well as in LPS-administrated condition as compared to successful pregnancy controls on days 12 and 16 of pregnancy. Together, these findings indicate that the lower expression of A2V-ATPase corresponds with the dynamic changes in spleen cell populations of abortion-prone matings. This supports our hypothesis that A2V-ATPase participates in the establishment of a unique immune milieu that helps the fetus to survive and develop in the uterus until parturition. This research was supported by Clinical Laboratory, Rosalind Franklin University of Medical and Science. (poster)

Objective: To investigate lipopolysaccharide (LPS)-induced DNA damage in preimplanting embryonic and uterine cells during preimplantation period of pregnancy that may ultimately inhibit the process of implantation in mouse.Design: Animal study.Setting: Academic research environment.Animal(s): Sixty four Park strain female mice.Intervention(S): The "minimum dose" (MD) of LPS was injected intraperitoneally in the pregnant females oil day 0.5 of pregnancy, and individual embryos and uterine cells were assessed by comet assay on days 1.5, 2.5, 3.5, and 4.375 of the preimplantation period of pregnancy.Main Outcome Measure(s): Percentage of embryos and uterine cells with tail, mean comet tail length, percentage of fragmented DNA in tail.Result(s): Significantly higher numbers of embryos with higher mean comet tail length and percentage of fragmented DNA in tail were observed in the LPS-treated compared with control animals as the period of pregnancy approaches the stage of implantation. At the same time, DNA damage was also significantly higher in the uterine cells of LPS-treated compared with control animals.Conclusion(s): The MD of LPS can induce DNA damage in the preimplantation-stage embryos and uterine cells, which causes poor embryonic development and improper preparation of uterine horns during the preimplantation period of pregnancy, which may ultimately inhibit the process of implantation in mouse. (Fertil Steril (R) 2009; 91:2095-103. (C)2009 by American Society for Reproductive Medicine.)

Endotoxins (lipopolysaccharides: LPS) are the main antigenic components of these bacteria and can trigger the immune system through the secretion of proinflammatory cytokines. Many of the cytokines are known to be involved in the process of embryonic development and implantation of the blastocyst on to a receptive endometrium for successful pregnancy to occur. In earlier studies, in a mouse model it was shown that the normal pattern of expression for IL-1, TNF-alpha, CSF-1 etc., in the embryos and uterine horns are altered when the animals are exposed to a 'minimum dose' (MD) of LPS. The current study investigates whether LPS can cause apoptosis in the preimplantation stage embryos as an end effect. Material and Methods: 5 mu g LPS were injected per mouse (weighing approximate to 20 gr) on day 0.5 of pregnancy. The degree of apoptosis induced by the MD of LPS was assessed by comet assays on individual preimplantation stage embryos.Results: MD of LPS was found to cause cell deaths in more than 88% of the embryos by the time they reach to the stage of implantation.Discussion: We conclude that LPS can cause implantation failure and pregnancy loss due to apoptosis of blastomeres during embryonic development to the blastocysts in vivo.