Increased Anti-HSP60 and Anti-HSP70 Antibodies in Women with Unexplained Recurrent Pregnancy Loss.

　Vascular dysfunction has been reported in women with recurrent pregnancy loss (RPL). We investigated the severity of vascular dysfunction in non-pregnant women with RPL and its correlation with anti-heat shock protein (HSP) antibodies that are known to induce arteriosclerosis. We measured the serum anti-HSP60 antibodies, anti-HSP70 antibodies, and anti-phospholipid antibodies (APA) in 68 women with RPL and 29 healthy controls. Among the women with RPL, 14 had a diagnosis of antiphospholipid syndrome (APS), and in the remaining 54, the causes for RPL were unexplained. Compared to the controls, the brachial-ankle pulse wave velocity (baPWV), carotid augmentation index (cAI), and uterine artery pulsatility index (PI) were all significantly higher in the women with both APS and unexplained RPL. Compared to the controls, the anti-HSP60 antibody levels were significantly higher in the APA-positive group of women with unexplained RPL, and the anti-HSP70 antibody levels were significantly higher in APS and APA-positive group of women with unexplained RPL. However, the anti-HSP60 and anti-HSP70 antibody levels did not correlate with the values of baPWV or cAI. Our results demonstrated anti-HSP60 and anti-HSP70 antibodies are increased in women with unexplained RPL. Further studies are needed to elucidate the roles of anti-HSP antibodies and their pathophysiology in unexplained RPL.

Adverse effects of advanced glycation end products on embryonal development.

We studied the effects of advanced glycation end products (AGEs), which are known to accumulate in patients with diabetes, autoimmune diseases, or those who smoke, on embryonal development. Pronuclear (PN) embryos were obtained by flushing the fallopian tubes of rats after superovulation and mating. The cleavage rate and blastocyst yield were evaluated at 24, 72, 96, and 120 h of culture. Glyoxal, an AGE-forming aldehyde, suppressed embryonal development at every stage from PN to blastocyst in a concentration-dependent manner. The cleavage rate of the embryo was also signifi cantly decreased by treatment with glyoxal at concentrations of 1 mM or higher. The blastocyst yield was significantly decreased by treatment with glyoxal at concentrations of 0.5 mM or higher. N-acetyl-L-cysteine (L-NAC) at 1 mM significantly suppressed the glyoxal-induced embryonal toxicity. BSA-AGEs at 5 microg/ml or higher concentration signifi cantly reduced the cleavage rate and blastocyst yield compared to those for BSA-treated embryos. L-NAC at 1 mM significantly suppressed BSAAGE-induced embryonal toxicity. Because AGEs are embryo-toxic, AGE contamination may influence the pregnancy rate of in vitro fertilization and embryo transfer. AGEs, which are increased in women under pathological conditions, may also be involved in their infertility.

Impaired uterine perfusion associated with metabolic disorders in women with polycystic ovary syndrome.

BACKGROUND: Risk factors for cardiovascular disease, including chronic anovulation, obesity, hyperandrogenism, hyperinsulinemia, and dyslipidemia, are commonly observed in women with polycystic ovary syndrome (PCOS). We evaluated uterine perfusion and its correlation with clinical and biochemical parameters in women with PCOS. METHODS: We performed a pulsed Doppler study on uterine arterial blood flow in 25 women with PCOS and 45 control women with regular menstrual cycles. PCOS was diagnosed based on oligomenorrhea, polycystic ovaries determined by means of ultrasonography, and elevated luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratio. RESULTS: Women with PCOS had a significantly higher body mass index (BMI) and serum testosterone, and showed insulin resistance and dyslipidemia, including increased total cholesterol, triglyceride, low-density lipoprotein-cholesterol (LDL-C), and decreased high-density lipoprotein-cholesterol (HDL-C). The uterine arterial pulsatility index (PI) in women with PCOS was significantly higher than that in the control women during the follicular phase. The PI was correlated with BMI, LH/FSH ratio, or LDL-C/HDL-C ratio, whereas it was inversely correlated with the HDL-C level. Women with PCOS had reduced endometrial thickness and elevated uterine arterial PI in the luteal phase, in which implantation occurs. CONCLUSIONS: Elevation of uterine arterial blood flow resistance is associated with risk factors for cardiovascular events. Furthermore, the impaired uterine perfusion in the luteal phase may cause endometrial dysfunction in women with PCOS.

Use of artificial dermis and recombinant basic fibroblast growth factor for creating a neovagina in a patient with Mayer-Rokitansky-Kuster-Hauser syndrome.

Vaginal agenesis is an uncommon, but not rare, condition. Although there are many methods for creating a neovagina, the optimal treatment is unknown. An 18-year-old woman with Mayer-Rokitansky-Küster-Hauser syndrome received vaginoplasty with a modified Wharton procedure using an artificial dermis (atelocollagen sponge). From 10 days after the operation, the patient was administered human recombinant basic fibroblast growth factor (bFGF) spray to accelerate epithelialization on the neovagina. At 50 days after the operation, we confirmed histological squamous epithelialization of the vaginal epithelium. At 12 months after the operation, the neovagina was at least 3.5 cm in width and approximately 8 cm in length. In this case, use of artificial dermis and recombinant bFGF to create a neovagina was an easy, less invasive and useful method.

Nafamostat mesilate suppresses NF-kappaB activation and NO overproduction in LPS-treated macrophages.

Nafamostat mesilate (NM), a clinically used serine protease inhibitor, suppressed the overproduction of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) in RAW264.7 murine macrophages treated with lipopolysaccharide (LPS, 100 ng/ml); however, it had little effect on endothelial NOS (eNOS) in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assay (EMSA) revealed that LPS activated nuclear factor-kappaB (NF-kappaB) in RAW264.7 cells and that this activation was suppressed by nafamostat mesilate. Western blotting showed that nafamostat mesilate suppressed the phosphorylation and degradation of inhibitor kappaB-alpha (IkappaB-alpha), which holds NF-kappaB in the cytoplasm in an inactivated state. Our observations suggest that nafamostat mesilate is a candidate agent for various diseases such as ischemia-reperfusion, graft rejection, inflammatory diseases, and autoimmune diseases, in which iNOS and/or NF-kappaB are upregulated.

Increased plasma adrenomedullin in women with recurrent pregnancy loss.

OBJECTIVE: To evaluate vascular changes and uterine perfusion in women with recurrent pregnancy loss. METHODS: We measured plasma levels of adrenomedullin of 100 pregnant women in the midluteal phase of a nonpregnant cycle (control group: n = 62; recurrent pregnancy loss group: n = 38). We measured the pulsatility index (PI) in the uterine arteries by transvaginal pulsed Doppler ultrasonography at the same time. RESULTS: The plasma level of adrenomedullin in women with recurrent pregnancy loss (5.6 +/- 1.9, mean +/- standard deviation) was significantly higher (P >.001) than that in control women (3.6 +/- 1.7). Uterine arterial PI of women with recurrent pregnancy loss (2.70 +/- 0.47) was significantly higher (P >.001) than that in control women (2.09 +/- 0.39). Plasma level of adrenomedullin had a significant positive correlation with uterine arterial PI both in the control group (r =.58, P <.001) and in the recurrent pregnancy loss group (r =.78, P <.001). Both plasma adrenomedullin concentration (7.2 +/- 2.3) and uterine arterial PI (3.06 +/- 0.36) were significantly high in women with antiphospholipid antibodies. CONCLUSION: Plasma adrenomedullin may serve as a useful biochemical marker for recurrent pregnancy loss caused by impaired uterine perfusion.

Involvement of peroxynitrite in LPS-induced apoptosis of trophoblasts.

OBJECTIVE: To examine whether or not peroxynitrite was involved in trophoblastic apoptosis induced by a bacterial endotoxin, lipopolysaccharide (LPS). METHODS: Levels of nitrite/nitrate, stable metabolites of nitric oxide (NO), in culture medium of trophoblasts, were determined using Griess reagents. Trophoblastic apoptosis was identified morphologically and confirmed using in situ nick end labeling technique. The amount of nitrotyrosine, a footprint of peroxynitrite, was quantified by dot blotting. Statistical significance was determined by ANOVA. RESULTS: Treatment of trophoblasts with LPS leads to apoptosis accompanied by formation of NO and nitrotyrosine. Aminoguanidine, an inhibitor of NO synthase (NOS), reduced peroxynitrite formation and prevented apoptosis. Scavengers of peroxynitrite also prevented apoptosis in this culture model. CONCLUSION: Peroxynitrite was involved in trophoblastic apoptosis induced by LPS. Peroxynitrite scavengers or inhibitors of NOS may thus be candidate therapeutic agents for infectious diseases, which is associated with overproduction of NO and peroxynitrite.

Impaired uterine arterial blood flow in pregnant women with recurrent pregnancy loss.

OBJECTIVE: This study was undertaken to evaluate uterine perfusion, which regulates uterine receptivity, in women with recurrent pregnancy loss. METHODS: We evaluated the blood flow resistance in the uterine arteries of 104 pregnant women at 4 to 5 weeks' gestation by transvaginal pulsed Doppler ultrasonography (control group, n = 52; and recurrent pregnancy loss group, n = 52). Blood tests for antinuclear and antiphospholipid antibodies were also performed. RESULTS: The uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group. Women with antinuclear or antiphospholipid antibodies had an elevated pulsatility index in the uterine artery, which is prominent in women with recurrent pregnancy loss. Coagulopathy and vascular dysfunction caused by autoantibodies may impair uterine perfusion. However, the uterine arterial pulsatility index in the recurrent pregnancy loss group was significantly higher than that in the control group even among women without antinuclear antibodies or among women without antiphospholipid antibodies. This observation strongly suggests that the uterine artery pulsatility index may be an independent index for recurrent pregnancy loss. CONCLUSIONS: The introduction of pulsed Doppler ultrasonography has provided the means for noninvasive evaluation of uterine impedance and may identify patients with recurrent pregnancy loss associated with impaired uterine perfusion.

Bisphenol A stimulates NO synthesis through a non-genomic estrogen receptor-mediated mechanism in mouse endothelial cells.

Biological actions of bisphenol A (BPA), an environmental chemical, have not been fully elucidated. We studied effect of BPA on nitric oxide (NO) synthesis in the murine endothelial cell line, MSS31. BPA (1-100 microM) increased nitrite/nitrate, a stable metabolites of NO, levels in culture medium of MSS31. However, Western blotting showed that the level of endothelial NO synthase protein was not increased by 16 h of treatment with BPA (10 microM). ICI 182,780 (10 microM), an estrogen receptor (ER) antagonist, suppressed BPA-induced NO synthesis while actinomycin D (1 microg/ml), a transcription inhibitor, or cycloheximide (40 microM), a protein synthesis inhibitor, exhibited no effect on BPA-induced NO synthesis. These results indicate that BPA stimulates NO synthesis through a non-genomic ER-mediated mechanism. Short-term effects of BPA on NO synthesis were weak but similar to 17beta-estradiol.

A long-term transdermal nitric oxide donor improves uteroplacental circulation in women with preeclampsia.

OBJECTIVE: To determine the effects of long-term transdermal administration (range, 4-30 days; mean +/- SD, 11.1+/-7.2 days) of isosorbide dinitrate, a nitric oxide donor, in preeclamptic women. METHODS: We studied uterine and fetoplacental circulation of 12 preeclamptic women with oligohydramnios and an elevated pulsatility index in the uterine arteries. RESULTS: Transdermal isosorbide dinitrate significantly suppressed the blood pressure of patients. Pulsed Doppler ultrasonography revealed that the average pulsatility index in the uterine arteries was significantly reduced by treatment with isosorbide dinitrate (P < .003). The average pulsatility index in the umbilical artery was also significantly reduced (P < .004). Furthermore, the size of the amniotic fluid pocket increased approximately 4-fold by treatment with isosorbide dinitrate. CONCLUSIONS: Long-term transdermal administration of isosorbide dinitrate improves fetoplacental circulation and may be effective therapy for avoiding maternal hypertension and oligohydramnios in some preeclamptic women.

The biological effects of antiadhesion agents on activated RAW264.7 macrophages.

The objective of this study is to determine the biological effects of various antiadhesion agents on macrophages, which play an essential role in wound healing and adhesion. To determine these effects, RAW264.7 macrophages were activated with lipopolysaccharide in the presence of antiadhesion agents: oxidized regenerated cellulose (oxyC), sodium hyaluronate (HA), dexamethasone (Dex), or chondroitin sulfate (CS). The release of nitric oxide (NO), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), or matrix metalloproteinases (MMPs) from RAW264.7 was measured. We found that oxyC reduced the release of NO, IL-6, MMP-2, and MMP-9, whereas it enhanced the release of VEGF. HA reduced the release of MMP-2, whereas it enhanced the release of VEGF and NO. HA exhibited no significant effect on the release of IL-6 or MMP-9. Dex reduced the release of NO, VEGF, IL-6, MMP-2, and MMP-9. CS reduced the release of VEGF, IL-6, and MMP-2, although it had no significant effect on the release of NO and MMP-9. Antiadhesion agents, which have been clinically used as physical barriers, modulated the functions of macrophages.